U.S. patent number 5,763,599 [Application Number 08/693,217] was granted by the patent office on 1998-06-09 for nucleoside derivatives with photolabile protective groups.
This patent grant is currently assigned to Wolfgang Pfleiderer. Invention is credited to Heiner Giegrich, Wolfgang Pfleiderer.
United States Patent |
5,763,599 |
Pfleiderer , et al. |
June 9, 1998 |
Nucleoside derivatives with photolabile protective groups
Abstract
The invention relates to nucleoside derivatives having
photolabile protective groups of the general formula (I) ##STR1##
in which R.sup.1 .dbd.H, NO.sub.2, CN, OCH.sub.3, halogen or alkyl
or alkoxyalkyl having 1 to 4 C atoms R.sup.2 .dbd.H, OCH.sub.3
R.sup.3 .dbd.H, F, Cl, Br, NO.sub.2 R.sup.4 .dbd.H, halogen,
OCH.sub.3, or an alkyl radical having 1 to 4 C atoms R.sup.5 .dbd.H
or a usual functional group for preparing oligonucleotides R.sup.6
.dbd.H, OH, halogen or XR.sup.8, where X.dbd.O or S and R.sup.8
represents a protective group usual in nucleotide chemistry,
B=adenine, cytosine, guanine, thymine, uracil,
2,6-diaminopurin-9-yl, hypoxanthin-9-yl, 5-methylcytosin-1-yl,
5-amino-4-imidazolcarboxamid-1-yl, or
5-amino-4-imidazolcarboxamid-3-yl, where in the case of B=adenine,
cytosine or guanine, the primary amino function optionally exhibits
a permanent protective group. These derivatives may be used for the
light-controlled synthesis of oligonucleotides on a DNA chip.
Inventors: |
Pfleiderer; Wolfgang (Konstanz
D-78464, DE), Giegrich; Heiner (Konstanz,
DE) |
Assignee: |
Pfleiderer; Wolfgang (Konstanz,
DE)
|
Family
ID: |
6536071 |
Appl.
No.: |
08/693,217 |
Filed: |
August 9, 1996 |
PCT
Filed: |
December 15, 1995 |
PCT No.: |
PCT/EP95/04976 |
371
Date: |
August 09, 1996 |
102(e)
Date: |
August 09, 1996 |
PCT
Pub. No.: |
WO96/18634 |
PCT
Pub. Date: |
June 20, 1996 |
Foreign Application Priority Data
|
|
|
|
|
Dec 16, 1994 [DE] |
|
|
44 44 996.8 |
|
Current U.S.
Class: |
536/55.3;
536/25.3; 536/26.1 |
Current CPC
Class: |
C07H
19/10 (20130101); C07H 19/06 (20130101); C07H
19/20 (20130101); C07H 21/00 (20130101); C07H
19/16 (20130101); C07H 19/04 (20130101); Y02P
20/55 (20151101) |
Current International
Class: |
C07H
19/20 (20060101); C07H 21/00 (20060101); C07H
19/16 (20060101); C07H 19/06 (20060101); C07H
19/10 (20060101); C07H 19/00 (20060101); C07H
19/04 (20060101); C07H 001/02 (); C07H 019/10 ();
C07H 019/20 (); C07H 021/00 () |
Field of
Search: |
;536/26.1,55.3,25.3 |
Foreign Patent Documents
Other References
Proc. Natl. Acad. Sci. USA, Bd. 91, May 1 1994, pp. 5022-5026,
XP002003499, A.C. Pease et al.: "Light-Generated Oligonucleotide
Arrays For Rapid DNA Sequence Analysis". .
W. Pfleiderer et al.: "New Protecting Groups In Nucleoside and
Nucleotide Chemistry" in: Biophosphates and Their
Analogues-Synthesis, Structure, Metabolism and Activity, 1987, pp.
133-142, K.S. Bruzik and W.J. STEC, XP002003501. .
J. Org. Chem., Bd. 60, Mar. 10, 1995, pp. 1116-1117, XP002003500,
M.C. Pirrung and J-C. Bradley: "Dimethoxybenzoin Carbonates:
Photochemically-Removable Alcohol Protecting Groups Suitable For
Phosphoramidite-Based DNA Synthesis"..
|
Primary Examiner: Kunz; Gary L.
Attorney, Agent or Firm: Felfe & Lynch
Claims
We claim:
1. A nucleoside derivative having a photolabile protective group,
of the general formula ##STR11## wherein R.sup.1 .dbd.H, N.sub.2,
CN, OCH.sub.3, halogen or alkyl or akoxyalkyl having 1 to 4 C
atoms;
R.sup.2 .dbd.H or OCH.sub.3 ;
R.sup.3 .dbd.H, F, Cl, Br or NO.sub.2 ;
R.sup.4 .dbd.H, halogen, OCH.sub.3, or an alkyl radical having 1 to
4 C atoms;
R.sup.5 .dbd.H or a phosphoramidite group;
R.sup.6 .dbd.H, OH, halogen or XR.sup.8, where X.dbd.O or S, and
R.sup.8 is a protective group;
B=adenine, cytosine, guanine, thymine, uracil,
2,6-diaminopurin-9-yl, hypoxanthin-9-yl, 5-methylcytosin-1-yl,
5-amino-4-imidazolcarboxamid-1-yl or
5-amino-4-imidazolcarboxamid-3-yl, with the proviso that when B is
adenine, cytosine or guanine, the primary amino function optionally
exhibits a protective group.
2. The nucleoside derivative of claim 1 wherein each of R.sup.1,
R.sup.2, and R.sup.3 are H and R.sup.4 is halogen, OCH.sub.3 or
C.sub.1 -C.sub.4 alkyl.
3. The nucleoside derivative of claim 1 wherein R.sup.2 is
OCH.sub.3 and R.sup.3 is H.
4. The nucleoside derivative of claim 1 wherein R.sup.4 is a methyl
radical.
5. The nucleoside derivative of claim 1 wherein R.sup.5 is a
phosphoramidite group of the formula: ##STR12## the R.sup.7 groups
are the same or different and are linear or branched C.sub.1
-C.sub.4 alkyl radicals.
6. The nucleoside derivative of claim 5 wherein R.sup.7 is ethyl or
isopropyl radical.
7. The nucleoside derivative of claim 1 wherein R.sup.6 is an
XR.sup.8 group and R.sup.8 is an alkyl, alkenyl, acetal, or silyl
ether protective group in the case of X.dbd.O, or an alkyl
protective group in the case of X.dbd.S.
8. The nucleoside derivative of claim 7 wherein R.sup.6 is an
O-methyl or O-ethyl radical, an O-allyl radical, an
O-tetrahydropyranyl or O-methoxytetrahydropyranyl radical, or an
O-t-butyldimethylsilyl radical.
9. The nucleoside derivative of claim 1 wherein B is adenine,
cytosine, or guanine, and a phenoxyacetyl or dimethylformamidino
radical is used as a protective group.
10. The nucleoside derivative of claim 1 wherein B is adenine and a
benzoyl or p-nitrophenylethoxycarbonyl-(p-NPEOC) radical is used as
a protective group.
11. The nucleoside derivative of claim 1 wherein B is guanine and
an isobutyroyl or a p-NPEOC radical is used as a protective
group.
12. The nucleoside derivative of claim 1 wherein B is cytosine and
benzoyl is used as a protective group.
13. The nucleoside derivative of claim 1 wherein B is cytosine and
p-NPEOC is used as a protective group.
14. The nucleoside derivative of claim 1 wherein at least one of
R.sup.1 and R.sup.6 is fluorine, chlorine, or bromine.
15. A method of preparing a nucleoside derivative of claim 1
comprising:
(a) forming a chlorocarbonic acid ester by reacting, with a
phosgene derivative, an alcohol of the general formula ##STR13##
(b) reacting the chlorocarbonic acid ester formed in step a) with a
nucleoside of the general formula ##STR14## (c) purifying the
resulting nucleoside possessing a 5'-O-photolabile protecting
group.
16. The method of claim 15 further comprising:
(c) introducing a phosphoramidite group ##STR15## in the 3'
position of said nucleoside derivative with R.sup.5 .dbd.H.
17. The method of claim 15 wherein step (a) is performed in a
non-polar organic solvent at a temperature of from -20.degree. C.
to +25.degree. C.
18. The method of claim 15 where in step (a), a two-fold to
five-fold excess of said phosgene derivative, relative to the
alcohol, is used.
19. The method of claim 17 wherein the non-polar organic solvent is
toluene or THF.
20. The method of claim 17 wherein the concentration of the alcohol
component is 0.1 to 10.0 mol per 10 ml solvent.
21. The method of claim 15 wherein step (b) is performed at a
temperature of from -60.degree. C. to +25.degree. C. in a solvent
mixture comprising dichloromethane and a polar organic solvent
optionally in the presence of a base.
22. The method of claim 21 wherein the polar organic solvent is DMF
or pyridine.
23. The method of claim 21 wherein the solvent mixture in step (b)
is comprised of dichloromethane and DMF, and at least one base
selected from the group consisting of pyridine, triethylamine, and
ethyl di-isopropylamine.
24. The method of claim 15 wherein the molar ratio of nucleoside to
chlorocarbonic acid ester is 1:1 to 1:2.
25. The method of claim 23 wherein in step (b), the nucleoside
dissolved in pyridine or DMF/base is charged and a solution of the
chlorocarbonic acid ester in dichloromethane is added dropwise at
the respective reaction temperature.
26. The method of claim 21 wherein the concentration of the
nucleoside in said solvent mixture in step (b) is 0.1 to 3.0 mmol
per 10 ml solvent.
27. The method of claim 16 wherein the introduction of said
phosphoramidite group (step c) is performed at a temperature of
from 0.degree. to 25.degree. C. by reacting said nucleoside
derivative with R.sup.5 .dbd.H with a corresponding phosphine in
the presence of H tetrazole as an activator in a solvent mixture
composed of dichloromethane and acetonitrile.
28. The method of claim 23 wherein the volume ratio of
dichloromethane to pyridine or the ratio of DMF to pyridine is 1:1
to 3:1.
29. A method for synthesizing oligonucleotides in solution or on a
solid phase support comprising covalently adding a nucleotide to an
oligonucleotide chain wherein the improvement is the substitution
of the 5'-O-photolabile nucleosides of claim 1 for the traditional
5'-protected nucleotide of the art of oligonucleotide synthesis
combined with the deprotecting of said 5'-photolabile protecting
groups with light of the appropriate wavelength.
30. The method of claim 29 wherein the oligonucleotides are
synthesized on a solid phase.
Description
This case is filed under 35 USC 371 as the U.S. stage of
PCT/EP95/04976 filed Dec. 15, 1995.
BACKGROUND OF THE INVENTION
The present invention relates to nucleoside derivatives with
photolabile protective groups and a method for their
preparation.
Photolabile protective groups for the hydroxy and phosphate
functions in nucleosides and nucleotides are of particular interest
since they are suitable for example for light-controlled parallel
syntheses of oligonucleotides on a solid carrier (cf. S.P.A. Fodor
et al. Science 1991, 251, p. 767 et seq.). They enable the
production of so-called DNA chips (i.e. carrier plates on the
surface of which a great number of many different oligonucleotides
are arranged), which in turn are required in molecular biology for
a rapid DNA sequence analysis.
In the prior art, the o-nitrobenzyl group and its derivatives have
so far mainly been used as photolabile protective groups in
nucleoside and nucleotide chemistry (cf. V.N.R. Pillai, Org.
Photochem. 1987, 9, p. 225 et seq. and J. W. Walker et al., J. Am.
Chem. Soc. 1988, 110, p. 7170 et seq.). The slow and partially only
incomplete deprotection of the corresponding nucleoside and
nucleotide derivatives proved to be a particular disadvantage of
these protective groups. Furthermore, undesirable by-products in
the form of toxic nitrosophenyl compounds are also obtained to some
extent during the cleavage of the o-nitrobenzyl compounds.
According to the article by W. Pfleiderer et al. in "Biophosphates
and Their Analogues--Synthesis, Structure, Metabolism and
Activity", Elsevier Science Publishers B.V. (Amsterdam) 1987, p.
133 et seq., the 2-(o-nitrophenyl)ethyl group which however is
introduced solely as a protective group in the base part,
particularly in O.sup.6 position of a guanosine, was also
recommended as another photolabile protective group for
nucleosides. The same publication also describes the
p-nitrophenylethoxycarbonyl (NPEOC) and the
2,4-dinitrophenylethoxycarbonyl (DNPEOC) groups both as protective
groups for the amino function and for the hydroxyl functions in the
sugar part, though elimination of these groups has been carried out
solely by means of base-catalyzed .beta.-elimination.
The present invention therefore has as its object to develop
nucleoside derivatives with photolabile protective groups for the
5-OH function in the sugar part, which derivatives do not exhibit
the named disadvantages of the prior art, but can be deprotected
comparatively quickly, quantitatively and without the formation of
undesirable by-products.
THE INVENTION
This object was solved according to the invention by means of
nucleoside derivatives of the general formula (I). Surprisingly, it
was in fact shown that the protective groups according to the
invention can be eliminated much more quickly and completely than
for example the o-nitrobenzyl groups. It has so far not been
possible to find any undesirable by-products during deprotection,
which had not been predictable either.
The nucleoside derivatives according to the invention are
represented by the following general formula (I): ##STR2## wherein
the radicals R.sup.1, R.sup.2 and R.sup.3 at the phenyl ring may
have the following meaning:
R.sup.1 .dbd.H, NO.sub.2, CN, OCH.sub.3, halogen or alkyl or
alkoxyalkyl with 1 to 4 C atoms
R.sup.2 .dbd.H, OCH.sub.3
R.sup.3 .dbd.H, F, Cl, Br, NO.sub.2
According to a preferred embodiment, R.sup.3 .dbd.H, if R.sup.2
.dbd.OCH.sub.3.
The radical R.sup.4 located on the C.sub.2 atom of the
o-nitrophenylethyl group may be either H, halogen, OCH.sub.3 or an
alkyl radical with 1 to 4 C atoms. The alkyl radical may in this
regard be linear or branched, substituted (particularly with one or
more halogen atoms) or unsubstituted as well as saturated or
unsaturated; the same also applies to the alkyl and alkoxyalkyl
radicals in R.sup.1. R.sup.4 preferably represents a methyl
radical. In the event that R.sup.4 .noteq.H, the substituents
R.sup.1, R.sup.2 and R.sup.3 at the phenyl ring are preferably
hydrogen radicals. Furthermore, in case that R.sup.2
.dbd.OCH.sub.3, R.sup.3 represents in particular a hydrogen
radical.
In this application, halogen consistently means F, Cl, Br, I and
preferably F, Cl or Br.
The nucleoside part of the compounds according to the invention is
composed of the usual D-ribofuranose or 2'-deoxyribofuranose units
and the pyrimidine (B=cytosine, thymine, uracil) or purine bases
(B=adenine, guanine). 2,6-diaminopurin-9-yl, hypoxanthin-9-yl,
5-methylcytosin-1-yl, 5-amino-4-imidazolcarboxamid-1-yl or
5-amino-4-imidazolcarboxamid-3-yl radicals can also be used as
bases.
The OH group(s) in the ribofuranoside or 2'-deoxyribofuranose part
may be free or protected, depending on demand. In this regard, the
known phosphoramidite groups such as ##STR3## have been successful
in protecting the 3' position, whereby the R.sup.7 groups can be
the same or different and mean linear or branched alkyl radicals
with 1 to 4 C atoms. They are preferably ethyl or isopropyl
radicals.
In the 2' position of the ribofuranoside part (position R.sup.6) a
free or a protected OH group may be present as well as a hydrogen
or halogen atom (particularly F, Cl, Br), whereby any protective
group (R.sup.8) common in nucleotide chemistry may be used. It is
possible to employ the conventional alkyl, alkenyl, acetal or silyl
ether protective groups for oxygen atoms (X.dbd.O). R.sup.6 may
also represent an S-alkyl group (X.dbd.S, R.sup.8 =alkyl).
Preferred examples for O-alkyl protective groups are O-methyl or
O-ethyl radicals; for O-alkenyl protective groups, O-allyl
radicals; for O-acetal protective groups, O-tetrahydropyranyl or
O-methoxytetrahydropyranyl radicals; and for O-silyl ether
protective groups, O-t-butyldimethylsilyl radicals.
According to a preferred embodiment, the pyrimidine or purine bases
with primary amino functions (e.g. adenine, cytosine and guanine)
may also contain preferably carbonyl-based permanent protective
groups. In this respect, phenoxyacetyl or dimethylformamidino
radicals are preferred which are possible for all three designated
bases. There are also special protective groups which are
introduced only in the case of certain bases. In the case of
adenine, for example, these are benzoyl or p-nitrophenyl
ethoxycarbonyl (p-NPEOC) radicals. In addition to the p-NPEOC
radicals, isobutyroyl protective groups can be introduced for
guanine. Finally, as well as the p-NPEOC radicals, benzoyl
protective groups are suitable for cytosine.
The preparation of the nucleoside derivatives according to the
invention can be conducted in two steps. In the first step a), an
alcohol of the general formula (II) ##STR4## in which R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 have the above-identified meaning, is
reacted with a phosgene derivative, preferably in a nonpolar
organic solvent at temperatures between -20.degree. and +25.degree.
C. Diphosgene (trichloromethyl chloroformate) or triphosgene
(bis-trichloromethyl carbonate) can be used as a phosgene
derivative in addition to the preferred phosgene.
The alcohol component is known in most cases or can be analogously
produced according to known processes. In step a), toluene or THF
is preferably used as a nonpolar organic solvent. Although the
reaction components can be used in an approximately stoichiometric
ratio, the phosgene derivative is preferably used in a clear
excess, for example in a two-to five-fold molar excess, in relation
to the alcohol component. The alcohol component concentration can
also be varied within broad limits though it has proved
particularly advantageous to set this concentration to 0.1 to 10.0
mmol per 10 ml solvent.
The corresponding chlorocarbonic acid esters of the general formula
(IV) ##STR5## are obtained in this reaction (reaction duration
approx. 1 to 6 hours) with a good purity and in a high yield
(>90%).
The work-up of the corresponding products preferably occurs by
first removing the excess phosgene and solvent by distillation in
vacuo. The chlorocarbonic acid ester (IV) can then be reacted
without further work-up in step b) with the nucleosides of the
general formula (III) ##STR6## wherein R.sup.5, R.sup.6 and B are
defined as above.
The reactions are preferably carried out in a solvent mixture
consisting of dichloromethane and a polar organic solvent
optionally in the presence of a base at temperatures between
-60.degree. and +25.degree. C. In this regard, DMF or pyridine are
preferably used as a polar organic solvent, whereby in the case of
pyridine, no additional base is necessary. But if
dichloromethane/DMF solvent mixtures are used, the addition of a
base such as pyridine, triethylamine or ethyl diisopropylamine is
recommended in order to scavenge the protons released during the
reaction. The mixture ratio of dichloromethane to pyridine or DMF
is not critical either, although 1 to 3 parts by volume
dichloromethane per part by volume pyridine or DMF are preferably
used.
According to a preferred embodiment, the corresponding nucleoside
(III) dissolved in pyridine or DMF/base is charged and a solution
of the chlorocarbonic acid ester in dichloromethane is added
dropwise at the respective reaction temperature. The molar ratio of
nucleoside to chlorocarbonic acid ester can be adjusted according
to the stoichiometry to approx. 1:1. Nevertheless, an excess of
chlorocarbonic acid ester is preferably used, this amount being
such that the molar ratio of nucleoside to chlorocarbonic acid
ester is 1:1 to 1:2. Finally, the concentration of the nucleoside
in the solvent mixture can also be varied within broad limits,
though it is preferably set to 0.1 to 3.0 mmol per 10 ml
solvent.
Once the reaction has been completed (reaction time approx. 1 to 5
hours), the nucleoside derivatives according to the invention can
be isolated or purified according to known methods, such as
dilution with dichloromethane, removing any salts by washing with
water, drying of the organic phase, concentration of the solution
or crystallization and subsequent silica gel chromatography. The
corresponding nucleoside derivatives can be obtained in this manner
with a high purity and in good yields (60 to 85%)
According to a preferred embodiment and following on from reaction
step b), the phosphoramidite group ##STR7## can be introduced into
the 3' position of the nucleoside derivatives with R.sup.5 .dbd.H
according to known methods. This reaction with the corresponding
phosphines usually takes place in the presence of 1H tetrazole as
an activator in a solvent mixture composed of dichloromethane and
acetonitrile at temperatures between 0.degree. and 25.degree. C.
The phosphine is preferably used in a two- to three-fold molar
excess whereas the molar ratio of phosphine to 1H tetrazole is set
to 3: approx. 1.0. The quantitative ratio of dichloromethane to
acetonitrile is not very critical and is preferably 1:1 to 4:1.
After the reaction has taken place (approx. 10 to 20 h), the
corresponding nucleoside can be worked up as described in step
b).
As irradiation experiments with polychromatic light having a
wavelength of >289 nm prove, the nucleosides according to the
invention can be deprotected very quickly (t.sub.0.5 =1 to 7 min)
and extensively (yields of up to 97%), thus satisfying the special
requirements expected of the protective group's photolability to an
excellent degree.
On account of these special properties, the nucleosides according
to the invention are extremely suitable for the preparation of
oligonucleotides by cleaving the protective groups in a
light-controlled manner, particularly on solid carrier plates.
The following examples are intended to illustrate the invention in
greater detail.
EXAMPLE 1
a) 2-(2-nitrophenyl)ethanol [1, 2]
KOH (21 mg, 0.37 mmol) was added to a mixture of o-nitrotoluene
(9.2 g, 67 mmol) and paraformaldehyde (0.8 g, 25 mmol) in DMSO (10
ml, synthesis quality, additionally dried for 2 d over molecular
sieve 4 .ANG.) which was then stirred for 2.5 h at 95.degree. C.
The solvent was removed and the residue purified by column
chromatography (SiO.sub.2, 20.times.3 cm, solvent: toluene 750 ml,
toluene/EtOAc 10:1 500 ml, 7:1 500 ml, 5:1 500 ml).
2-(2-nitrophenyl)ethanol (2.33 g, 21%) was obtained as a yellow
oil.
R.sub.f (SiO.sub.2, toluene/EtOAc 10:1) 0.21
UV (MeOH) .lambda..sub.max [nm] (log .epsilon.): 205 (4.07), 256
(3. 65), 348 (shoulder, 2.56)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.93 (dd, 1 arom. H, ortho to
NO.sub.2); 7.49 (m, 3 arom. H); 3.96 (t, .alpha.-CH.sub.2); 3.17
(t, .beta.-CH.sub.2), 1.72 (s, OH)
C.sub.8 H.sub.9 NO.sub.3 (167.2)
Literature
[1] G. M. Bennet, M. M. Hafez, J. Chem. Soc. 1941, 287
[2] E. Uhlmann, W. Pfleiderer, Helv. Chim. Acta 1981, 64, 1688
b) 2-(2-nitrophenyl)ethoxycarbonyl chloride
Phosgene was introduced into a solution of 2-(2-nitrophenyl)ethanol
(5.2 g, 31 mmol) in THF (20 ml, dist. over CaH.sub.2) at room
temperature under stirring. After 1.5 h, the excess phosgene and
the solvent were removed by distillation in a high vacuum.
2-(2-nitrophenyl)ethoxycarbonyl chloride (6.69 g, 94%) was obtained
as a yellow oil.
R.sub.f (SiO.sub.2, CHCl.sub.3) 0.84
UV(CH.sub.3 CN), .lambda..sub.max [nm] (log .epsilon.): 202 (4.12),
218 (shoulder, 3.74); 256 (3.70); 298 (shoulder, 3.16); 346
(shoulder, 2.59)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.99 (dd, H--C(3)); 7.48 (m, 3
arom. H); 4.62 (t, .alpha.-CH.sub.2); 3.31 (t,
.beta.--CH.sub.2)
Anal. calcd. for C.sub.9 H.sub.8 ClNO.sub.4 (229.62): C 47.08, H
3.51, N 6.10; found: C 47.30, H 3.70, N 6.00
c) 5'-O-(2-(2-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (1 g, 4.13 mmol) was co-evaporated with pyridine
(2.times.10 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.) dissolved in pyridine (10 ml, see above)
and cooled down to -30.degree. C. A solution of
2-(2-nitrophenyl)ethoxcarbonyl chloride (1.45 g, 6.31 mmol) was
added dropwise thereto for 10 min. After stirring for a further 4 h
50 min in conditions of i-PrOH/N.sub.2 cooling (-30.degree. to
-15.degree. C.), the mixture was diluted with CH.sub.2 Cl.sub.2
(150 ml) and washed with H.sub.2 O (150 ml). The aqueous phases
were post-extracted with CH.sub.2 Cl.sub.2 (2.times.150 ml). The
combined organic phases were dried over MgSO.sub.4, filtered,
concentrated under reduced pressure and co-evaporated using toluene
(5.times.20 ml) and CH.sub.2 Cl.sub.2 (2.times.20 ml). The crude
product was purified by column chromatography (SiO.sub.2,
15.times.3.5 cm, solvent: CH.sub.2 Cl.sub.2 1300 ml, CH.sub.2
Cl.sub.2 /acetone 20:1 1200 ml, 10:1 600 ml, 8:1 500 ml, 5:1 500
ml, 4:1 500 ml, 2:1 750 ml, 1:1 500 ml).
5'-O-(2-(2-nitrophenyl)ethoxycarbonyl)thymidine (1.15 g, 64%) was
obtained as a colourless solid.
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.46
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.31), 262
(4.12), 334 (shoulder, 2.64)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.92 (s (br), NH); 7.96 (dd,
(H--C(3) of NPEOC); 7.55 (t, 1 arom. H of NPEOC); 7.42 (m, H--C(6)
of thymine, 2 arom. H of NPEOC); 6.35 (t, H--C(1')); 4.44 (m,
H--C(3'), 2.times.H--C(5'), .alpha.-CH.sub.2 of NPEOC); 4.15 (q,
H--C(4'); 3.34 (m, .beta.-CH.sub.2 of NPEOC); 2.89 (d, OH--C(3'));
2.41 (m, H--C(2')); 2.22 (m, H--C(2')); 1.85 (s, CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.21 N.sub.3 O.sub.9 (435.39): C
52.41, H 4.86, N 9.65; found: C 52.07, H 5.15, N 9.65
EXAMPLE 2
a) 2-(2,6-dinitrophenyl)ethanol [1]
A solution of potassium tertiary-butylate (1.8 g, 8 mmol) in
tert.-butanol (20 ml, synthesis quality, 99%) was added to
2,6-dinitrotoluene (18.2 g, 0.1 mol, dried for 3 d in high vacuum
over silica gel) and paraformaldehyde (3 g, 0.1 mol) in DMSO (50
ml, synthesis quality, additionally dried for 2 d over molecular
sieve 4 .ANG.). After the addition of the potassium
tertiary-butylate solution, a color change from yellow to deep
violet occurred. The solution was stirred for 5 min at room
temperature and for 10 min at 70.degree. C. (oil bath temperature).
It was then left to cool to room temperature, and the mixture was
neutralized with conc. HCl, and diluted with H.sub.2 O (300 ml)
before NaCl was added until the mixture was saturated. The aqueous
phase was extracted with EtOAc (3.times.500 ml). The combined
organic phases were washed with a saturated NaCl solution (300 ml),
dried over Na.sub.2 SO.sub.4, filtered and concentrated under
reduced pressure. The crude product (24.3 g) was dissolved in a
little EtAOc at boiling temperature, mixed with petroleum ether
(henceforth referred to as PE) (100 ml) and crystallized inside a
freezer. The precipitate was filtered by suction and purified by
column chromatography (20.7 g crude product, 300 g SiO.sub.2,
18.times.6.5 cm, solvent: toluene/EtOAc 5:1, 4:1, 3:1). Mixed
fractions were concentrated under reduced pressure and purified by
renewed column chromatography (200 g SiO.sub.2, 20.times.5.3 cm,
solvent: toluene/EtOAc 7:1). After concentrating the pooled pure
product fractions in a rotating evaporator under reduced pressure,
2-(2,6-dinitrophenyl)ethanol (13.6 g, 64%) was obtained as a yellow
solid.
R.sub.f (SiO.sub.2, toluene/EtOAc 9:1) 0.21
Melting point: 69.degree. to 70.degree. C. (Lit. [1]: 69.degree.
C.)
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 203 (4.20), 231
(4. 00), 280 (shoulder, 3.17), 327 (shoulder, 2.80)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.00 (d, J=8.1, H--C(3),
H--C(5)); 7.57 (t, J=8.1, H--C(4)); 3.97 (q, .alpha.-CH.sub.2);
3.33 (t, .beta.-CH.sub.2); 1.69 (t, OH)
Anal. calcd. for C.sub.8 H.sub.8 N.sub.2 O.sub.5 (212.16): C 45.29,
H 3.80, N 13.20; found: C 45.39, H 3.90, N 13.32
Literature
[1] N. S. Girgis, H. B. Cottam, R. K. Robins, J. Heterocycl. Chem.
1988, 25, 361
b) 2-(2,6-dinitrophenyl)ethoxycarbonyl chloride
A solution of 2-(2,6-dinitrophenyl)ethanol (4.08 g, 19.23 mmol) and
Et.sub.3 N (2.7 ml, 19.28 mmol) in THF (30 ml, dist. over
CaH.sub.2) was dropped in a solution, cooled to 0.degree. C., of
trichloromethyl chloroformate (3.81 g, 2.33 ml, 19.28 mmol) in THF
(10 ml, see above) for 20 min. This solution was stirred for 25 min
while cooling in an ice bath, and was then stirred for 1 h 45 min
at room temperature. The mixture was filtered over Celite.
Rewashing the filter cake with THF, removing the solvent and excess
reagent from the pooled filtrates by distillation and drying in a
high vacuum yielded 5.13 g 2-(2,6-dinitrophenyl)-ethoxycarbonyl
chloride (97%) as a light-brown solid.
R.sub.f (SiO.sub.2, CH.sub.2 Cl.sub.2) 0.76
melting point: 84.degree. to 85.degree. C.
UV(CH.sub.3 CN), .lambda..sub.max [nm] (log .epsilon.): 233 (4.02),
292 (shoulder, 3.11), 331 (shoulder, 2.82)
.sup.1 H-NMR (250 MHz, CDCl.sub.3) : 8.10 (d, J=8.2, H--C(3),
H--C(5)); 7.67 (t, J=8.1, H--C(4)); 4.67 (t, .alpha.-CH.sub.2);
3.50 (t, .beta.-CH.sub.2);
Anal. calcd. for C.sub.9 H.sub.7 ClN.sub.2 O.sub.6 (274.616): C
39.36, H 2.57, N 10.20; found: C 39.40, H 2.60, N 10.20
c) 5'-O-(2-(2,6-dinitrophenyl)ethoxycarbonyl)thymidine
Thymidine (1 g, 4.13 mmol) was co-evaporated with pyridine
(3.times.10 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (15 ml, see above)
and cooled down to -50.degree. C. A solution of
2-(2,6-dinitrophenyl)ethoxycarbonyl chloride (1.7 g, 6.19 mmol) in
CH.sub.2 Cl.sub.2 (15 ml, dist. over CaH.sub.2) was added dropwise
thereto for 1 h. After a further 3.5 h stirring in conditions of
i-PrOH/N.sub.2 cooling (-50.degree. to -20.degree. C.), the mixture
was diluted with CH.sub.2 Cl.sub.2 (50 ml) and washed with H.sub.2
O (50 ml). The aqueous phases were post-extracted with CH.sub.2
Cl.sub.2 (2.times.50 ml). The combined organic phases were dried
over Na.sub.2 SO.sub.4, filtered, concentrated under reduced
pressure and co-evaporated using toluene (3.times.50 ml). The crude
product (2.66 g) was treated with CH.sub.2 Cl.sub.2 /MeOH 2:1 (60
ml) at boiling temperature. The white precipitate obtained was
filtered by suction and recrystallized from MeOH (25 ml).
5'-O-(2-(2,6-dinitrophenyl)ethoxycarbonyl)thymidine (855 mg, 43%)
was obtained as a colorless solid. The combined filtrates were
concentrated under reduced pressure to a dry state and purified by
column chromatography (1.4 g crude product, 56 g SiO.sub.2
17.times.3 cm, CH.sub.2 Cl.sub.2 /MeOH 100:5 1240 ml, 100:7 105 ml,
100:10 330 ml). 5'-O-(2-(2,6-dinitrophenyl)ethoxcarbonyl)thymidine
(691 mg, 34.8%) was obtained as a colorless solid. The yield of
5'-O-(2-(2,6-dinitrophenyl)ethoxycarbonyl)thymidine totalled 1.55 g
(78%)
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.41
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 206 (4.28), 244
(shoulder, 4.07), 256 (4.08) 355 (shoulder, 2.72)
.sup.1 H-NMR (250 MHz, DMSO-d.sup.6) : 11.32 (s, NH), 8.27 (d,
H--C(3), H--C(5)); 7.78 (t, H--C(4)); 7.40 (s, H--C(6) of thymine);
6.18 (t, H--C(1')); 5.44 (d, OH--C(3')); 4.37 (t,
.alpha.-CH.sub.2); 4.23 (m, H--C(3'), 2.times.H--C(5')); 3.91 (m,
H--C(4')); 3.29 (t, .beta.-CH.sub.2); 2.15 (m, 2.times.H--C(2'));
1.71 (s, CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.20 N.sub.4 O.sub.11 (480.386): C
47.51, H 4.20, N 11.66; found: C 47.40, H 4.15, N 11.57
EXAMPLE 3
a) 2-(2-fluoro-6-nitrophenyl)ethanol [1]
A solution of potassium tertiary-butylate (90 mg, 0.8 mmol) in
tert.-butanol (1 ml, synthesis quality, 99%) was added to
2-fluoro-6-nitrotoluene (776 mg, 5 mmol) and paraformaldehyde (150
mg, 5 mmol) in DMSO (2.5 ml, synthesis quality, additionally dried
for 2 d over molecular sieve 4 .ANG.). After the addition of the
potassium tertiary-butylate solution, a color change from yellow to
deep violet occurred. The solution was stirred for 5 min at room
temperature and for 30 min at 70.degree. C. (oil bath temperature).
It was then left to cool down to room temperature and neutralized
with a few drops of conc. HCl. The mixture was diluted with EtOAc
(30 ml) and washed with H.sub.2 O (20 ml). The aqueous phase was
post-extracted with EtOAc (2.times.20 ml). Drying the organic
phases over Na.sub.2 SO.sub.4, filtering and evaporating the
solvent under reduced pressure yielded the crude product (1.11 g)
which was purified by column chromatography (20 g SiO.sub.2,
12.times.2 cm, solvent: PE 40 ml, PE/EtOAc 10:1 110 ml, 8:1 270 ml,
6:1 210 ml, 5:1 60 ml, 4:1 50 ml).
2-(2-fluoro-6-nitrophenyl)ethanol (653 mg, 71%) was obtained as a
yellow solid.
R.sub.f (SiO.sub.2, toluene/EtOAc 9:1) 0.24
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.06), 251
(3.61), 294 (shoulder, 3.21)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.73 (m, 1 arom. H); 7.36 (m, 2
arom. H); 3.94 (t, .alpha.-CH.sub.2); 3.21 (dt, J=2.2, 6.5,
.beta.-CH.sub.2); 1.67 (s (br), OH)
Anal. calcd. for C.sub.8 H.sub.8 FNO.sub.3 (185.154): C 51.90, H
4.36, N 7.57; found: C 51.92, H 4.40, N 7.42
Literature
[1] Chem. Abstr. 1989, 110, P 75032 k
b) 2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl chloride
A solution of 2-(2-fluoro-6-nitrophenyl)ethanol (500 mg, 2.7 mmol)
and Et.sub.3 N (273 mg, 2.7 mmol, dist. over KOH) in THF (6.75 ml,
dist. over CaH.sub.2) was dropped into a solution, cooled to
0.degree. C., of trichloromethyl chloroformate (641 mg, 3.24 mmol)
in THF (6.75 ml, see above) for 5 min. It was stirred for 1 h while
cooling in an ice bath and then stirred for 1 h at room
temperature. The mixture was filtered over Celite. Rewashing the
filter cake with THF and removing the solvent and excess reagent
from the pooled filtrates by distillation at 30.degree. C. in a
high vacuum yielded 2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl
chloride (620 mg, 93%) as a light-brown oil.
R.sub.f (SiO.sub.2, PE/EtOAc 19:1) 0.25
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 204 (4.04), 251
(3. 67), 293 (shoulder, 3.23)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.83 (d, J=7.7, arom. H); 7.44
(m, 2 arom. H); 4.63 (t, .alpha.-CH.sub.2); 3.37 (dt, J=1.6, 6.4
.beta.-CH.sub.2)
Anal. calcd. for C.sub.9 H.sub.7 ClFNO.sub.4 (247.609): C 43.66, H
2.85, N 5.66; found: C 43.97, H 3.02, N 5.59
c) 5'-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (200 mg, 0.83 mmol) was co-evaporated with pyridine
(3.times.3 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (3 ml, see above)
and cooled to -600.degree. C. (i-PrOH/N.sub.2). A solution of
2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl chloride (280 mg, 1.13
mmol) in CH.sub.2 Cl.sub.2 (3 ml, dist. over CaH.sub.2) was added
dropwise thereto for 20 min. It was stirred in conditions of
i-PrOH/N.sub.2 cooling (-60.degree. to -150.degree. C.) for 3 h 40
min and then for 1 h without a cooling bath, whereby the
temperature was 0.degree. C. toward the end. The reaction mixture
was diluted with CH.sub.2 Cl.sub.2 (10 ml) and washed with H.sub.2
O (10 ml). The aqueous phase was post-extracted with CH.sub.2
Cl.sub.2 (3.times.10 ml). Drying of the organic phases over
Na.sub.2 SO.sub.4, filtering, evaporating the solvent under reduced
pressure and co-evaporating with toluene (3.times.10 ml) yielded
the crude product which was purified by column chromatography (20 g
SiO.sub.2 12.times.2 cm, solvent: CH.sub.2 Cl.sub.2 /MeOH 100:1 50
ml, 100:2 102 ml, 100:3 206 ml, 100:3.5 103 ml, 100:4 208 ml).
First
3',5'-bis-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine,
then 3'-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine and
finally 5'-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine
was eluted. After evaporating the solvent under reduced pressure
and drying in a high vacuum,
3',5'-bis-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine (27
mg, 5%) was obtained as pale yellow foam,
3'-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine (15 mg,
4%) was obtained as a colorless foam and
5'-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine (262 mg,
70%) was obtained as a colorless solid.
5'-O-(2-(2-fluoro-6-nitrophenyl)ethoxycarbonyl)thymidine
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.44
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.29), 261
(4.10),
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.09 (s (br), NH); 7.76 (m, 1
arom. H of FNPEOC); 7.41 (m, H--C(6) of thymine, 2 arom. H of
FNPEOC), 6.35 (t, H--C(1')); 4.45 (m, H--C(3'), 2.times.H--C(5'),
.alpha.-CH.sub.2 of FNPEOC); 4.14 (q, J=3.2, H--C(4')); 3.36 (m,
.beta.-CH.sub.2 of FNPEOC); 2.40 (m, H--C(2')); 2.23 (m, H--C(2'),
OH--C(3')); 1.85 (S, CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.20 FN.sub.3 O.sub.9 (453.379): C
50.34, H 4.45, N 9.27; found: C 50.22, H 4.49, N 9.18
EXAMPLE 4
a) 2-(2-chloro-6-nitrophenyl)ethanol [1, 2]
Triton B (2 ml, 35% in MeOH) was added to a mixture composed of
2-chloro-6-nitrotoluene (25 g, 146 mmol) and paraformaldehyde (1.9
g, 60 mmol) in DMSO (20 ml, synthesis quality, additionally dried
for 2 d over molecular sieve 4 .ANG.) and was stirred at 90.degree.
C. After 2 h, the reaction mixture was neutralized with a few drops
of conc. HCl, diluted with H.sub.2 O (50 ml) and extracted with
EtOAc (4.times.150 ml). The combined organic phases were dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
crude product was purified by sublimation in a high vacuum (p=0.06
torr, oil bath temperature 95.degree. C.).
2-(2-chloro-6-nitrophenyl)ethanol (8.01 g, 66%) was obtained in the
form of bright yellow crystals.
R.sub.f (SiO.sub.2, toluene/EtOAc 10:1) 0.36
melting point: 59.degree. to 61.degree. C.
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 212 (4.13), 248
(3.48), 294 (shoulder, 3.03), 336 (shoulder, 2.58)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.70 (dd, 1 arom. H); 7.62 (dd,
1 arom. H); 7.31 (t, H--C(4)); 3.94 (q, .alpha.-CH.sub.2); 3.26 (t,
.beta.-CH.sub.2); 1.70 (t, OH)
Anal. calcd. for C.sub.8 H.sub.8 ClNO.sub.3 (201.61): C 47.66, H
4.00, N 6.95; found: C 47.79, H 4.06, N 6.92
Literature
[1] T. Morimoto, I. Hashimoto, H. Yamaoka, Chem. Abstr. 1978, 88,
104880 v.
[2] Y. Tsuji, S. Kotachi, K.-T. Huh, Y. Watanabe, J. Org. Chem.
1990, 55, 580
b) 2-(2-chloro-6-nitrophenyl)ethoxycarbonyl chloride
Phosgene was introduced in a solution of
2-(2-chloro-6-nitrophenyl)ethanol (31 g, 154 mmol) in THF (190 ml,
dist. over CaH.sub.2) at room temperature under stirring. After 2.5
h the excess phosgene and the solvent were removed by distillation
in a high vacuum. 2-(2-chloro-6-nitrophenyl)ethoxycarbonyl chloride
(39.4 g, 97%) was obtained as a yellow oil.
R.sub.f (SiO.sub.2, CHCl.sub.3) 0.76
UV(CH.sub.3 CN), .lambda..sub.max [nm] (log .epsilon.): 211 (4.14),
253 (3.53), 300 (shoulder, 3.02)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.81 (dd, 1 arom. H); 7.69 (dd,
1 arom. H); 7.41 (t, H--C(4)); 4.63 (t, .alpha.-CH.sub.2); 3.46 (t,
.beta.-CH.sub.2)
Anal. calcd. for C.sub.9 H.sub.7 Cl.sub.2 NO.sub.4 (264.06): C
40.94, H 2.67, N 5.30; found: C 41.05, H 2.73, N 5.00
c) 5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (4 g, 16.5 mmol) was co-evaporated with pyridine
(3.times.40 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (48 ml, see above)
and cooled to -50.degree. C. (i-PrOH/N.sub.2). A solution of
2-(2-chloro-6-nitrophenyl)ethoxycarbonyl chloride (5.2 g, 19.8
mmol) in CH.sub.2 Cl.sub.2 (48 ml, dist. over CaH.sub.2) was added
dropwise thereto for 2 h. It was stirred for another 30 min while
being cooled in i-PrOH/N.sub.2 conditions (-50.degree. to
-30.degree. C.) The reaction mixture was diluted with CH.sub.2
Cl.sub.2 (100 ml) and washed with H.sub.2 O (100 ml). The aqueous
phase was post-extracted with CH.sub.2 Cl.sub.2 (2.times.100 ml).
Drying of the organic phases over Na.sub.2 SO.sub.4, filtering,
evaporating the solvent under reduced pressure and co-evaporating
with toluene (3.times.50 ml) yielded the crude product which was
purified by column chromatography (305 g SiO.sub.2, 22.times.5.9
cm, solvent: CH.sub.2 Cl.sub.2 200 ml, CH.sub.2 Cl.sub.2 /MeOH
100:2 1020 ml, 100:3 515 ml, 100:4 1560 ml, 100:5 850 ml, 100:6 318
ml, 100:8 324 ml, 100:9 654 ml). First a mixed fraction (1.4 g) of
3',5'-bis-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine and
3'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine, then a
mixed fraction (367 mg) of
3'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine and
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine, and
finally a pure fraction of
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine (6.21 g,
80%, colorless solid) were eluted.
3',5'-bis-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine
(1.175 g, 10%) was obtained as a pale yellow foam,
3'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine (203 mg,
3%) was obtained as a colorless foam and
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine (182 mg,
2% ) was obtained as colorless solid. The total yield of
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine was
therefore 6.392 g (82%).
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.53
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 210 (4.36), 263
(4.05)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.11 (s, NH); 7.74 (d, J=8.2, 1
arom. H of ClNPEOC); 7.67 (d, J=8.0, 1 arom. H of ClNPEOC), 7.39
(t, H--C(4) of ClNPEOC); 7.38 (s, H--C(6) of thymine); 6.36 (t,
H--C(1')); 4.44 (m, .alpha.-CH.sub.2 of ClNPEOC, H--C(3'),
2.times.H--C(5')); 4.15 (q, H--C(4')); 3.45 (t, .beta.-CH.sub.2 of
ClNPEOC); 2.38 (m, H--C(2')); 2.26 (m, H--C(2')), 2.18 (d, J=3.9,
OH--C(3')); 1.86 (s, CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.20 ClN.sub.3 O.sub.9 (469.83): C
48.57, H 4.29, N 8.94; found: C 48.53, H 4.34, N 8.91
EXAMPLE 5
a) 2-(2-bromo-6-nitrophenyl)ethanol [1]
A solution of potassium tertiary-butylate (90 mg, 0.8 mmol) in
tert-butanol (1 ml, synthesis quality, 99%) was added to
2-bromo-6-nitrotoluene (1.08 g, 5 mmol) and paraformaldehyde (150
mg, 5 mmol in DMSO (2.5 ml, synthesis quality, additionally dried
for 2 d over molecular sieve 4 .ANG.). After the addition of the
potassium tertiary-butylate solution, a color change from yellow to
deep violet occurred. It was stirred for 5 min at room temperature
and for 30 min at 70.degree. C. (oil bath temperature). The mixture
was then allowed to cool to room temperature and neutralized with a
few drops of conc. HCl. The mixture was diluted with EtOAc (30 ml)
and washed with H.sub.2 O (20 ml). The aqueous phase was
post-extracted with EtOAc (2.times.20 ml). Drying of the organic
phases over Na.sub.2 SO.sub.4, filtering and evaporating the
solvent under reduced pressure yielded the crude product (1.49 g)
which was purified by column chromatography (20 g SiO.sub.2,
20.times.2 cm, solvent: PE 45 ml, PE/EtAOc 10:1 110 ml, 8:1 180 ml,
7.5:1 340 ml, 6:1 140 ml). 2-(2-bromo-6-nitrophenyl)ethanol (867
mg, 70%) was obtained as a yellow solid.
R.sub.f (SiO.sub.2, toluene/EtOAc 9:1) 0.32
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 204 (4.18), 210
(shoulder, 4.16), 251 (3.48), 293 (shoulder, 3.07)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.82 (dd, J=1.1, 8.0, 1 arom.
H); 7.74 (dd, J=1.1, 8.2, 1 arom. H); 7.26 (m, H--C(4)); 3.96 (t,
.alpha.-CH.sub.2); 3.30 (t, .beta.-CH.sub.2); 1.75 (s (br), OH)
Anal. calcd. for C.sub.8 H.sub.8 BrNO.sub.3 (246.06): C 39.05, H
3.28, N 5.69; found: C 39.09, H 3.26, N 5.56
Literature
[1] Chem. Abstr. 1989, 110, P 75032 k
b) 2-(2-bromo-6-nitrophenyl)ethoxycarbonyl chloride
A solution of 2-(2-bromo-6-nitrophenyl)ethanol (500 mg, 2.03 mmol)
and Et.sub.3 N (206 mg, 2.03 mmol, dist. over KOH) in THF (5 ml,
dist. over CaH.sub.2) was dropped into a solution, cooled to
0.degree. C., of trichloromethyl chloroformate (442 mg, 2.23 mmol)
in THF (5 ml, see above) for 10 min. It was stirred for 5 min while
being cooled in an ice bath and for 1 h 45 min at room temperature.
The mixture was filtered over Celite. Rewashing the filter cake
with THF and removing the solvent and the excess reagent from the
pooled filtrates by distillation at 30.degree. C. in a high vacuum
yielded 2-(2-bromo-6-nitrophenyl)ethoxycarbonyl chloride (625 mg,
99.8%) as a light brown oil.
R.sub.f (SiO.sub.2, PE/EtOAc 19:1) 0.31
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (shoulder,
4.14), 211 (4.16), 254 (3.52), 297 (shoulder, 3.05)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.85 (2 arom. H); 7.33 (t,
H--C(4)); 4.62 (t, .alpha.-CH.sub.2); 3.47 (t, .beta.-CH.sub.2)
Anal. calcd. for C.sub.9 H.sub.7 BrClNO.sub.4 (308.515): C 35.04, H
2.29, N 4.54; found: C 35.50, H 2.58, N 4.50
c) 51-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (200 mg, 0.83 mmol) was co-evaporated with pyridine
(3.times.3 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (3 ml, see above)
and cooled to -60.degree. C. (i-PrOH/N.sub.2). A solution of
2-(2-bromo-6-nitrophenyl)ethoxycarbonyl chloride (354 mg, 1.15
mmol) in CH.sub.2 Cl.sub.2 (3 ml, dist. over CaH.sub.2) was added
dropwise thereto for 10 min. It was stirred for 3 h 40 min in
conditions of i-PrOH/N.sub.2 cooling (-60.degree. to -20.degree.
C.). The reaction mixture was diluted with CH.sub.2 Cl.sub.2 (10
ml) and washed with H.sub.2 O (10 ml). The aqueous phase was
post-extracted with CH.sub.2 Cl.sub.2. Drying of the organic phases
over Na.sub.2 SO.sub.4, filtering, evaporating the solvent under
reduced pressure and co-evaporating with toluene (4.times.10 ml)
yielded the crude product (552 mg). Crystallisation from a little
CH.sub.2 Cl.sub.2 and MeOH enabled
5'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine (163 mg,
38%) to be isolated as a colorless solid. The residue was purified
by column chromatography (18 g SiO.sub.2, 11.times.2 cm, solvent:
CH.sub.2 Cl.sub.2 /MeOH 100:1 50 ml, 100:3 103 ml, 100:4 208 ml,
100:5 52 ml). First
3',5'-bis-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine,
then 3'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine and
finally 5'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine
were eluted. The product fractions were concentrated under reduced
pressure and dried in a high vacuum.
3',5'-bis-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine (62
mg, 10%) was obtained as a colourless foam,
3'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine (8 mg, 2%)
was obtained as a colorless oil and
5'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine (151 mg,
35%) as a colorless solid. The total yield of
5'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine was
therefore 314 mg (73%).
5'-O-(2-(2-bromo-6-nitrophenyl)ethoxycarbonyl)thymidine
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.38
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 210 (4.37), 263
(4.06)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.35 (s, NH); 7.85 (d, 1 arom.
H of BNPEOC); 7.76 (d, 1 arom. H of BNPEOC), 7.38 (s, H--C(6) of
thymine); 7.30 (t, H--C(4) of BNPEOC, partially concealed under
CHCl.sub.3 -signal); 6.37 (t, H--C(1')); 4.45 (m, H--C(3'),
2.times.H--C(5'), .alpha.-CH.sub.2 of BNPEOC)); 4.15 (m, H--C(4'));
3.47 (t, .beta.-CH.sub.2 of BNPEOC); 2.41 (m, H--C(2'), OH--C(3'));
2.25 (m, H--C(2')); 1.86 (s, CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.20 BrN.sub.3 O.sub.9 (514.285): C
44.37, H 3.92, N 8.17; found: C 44.31, H 3.96, N 8.11
EXAMPLE 6
a) 2-(4-chloro-2-nitrophenyl)ethanol [1, 2, 3]
Triton B (4 ml, 35% in MeOH) was added to a mixture of
4-chloro-2-nitrotoluene (50 g, 291 mmol) and paraformaldehyde (3.8
g, 120 mmol) in DMSO (40 ml, synthesis quality, additionally dried
for 2 d over molecular sieve 4 .ANG.) and stirred for 2.5 h at
900.degree. C. It was neutralized with a few drops of conc. HCl,
diluted with H.sub.2 O (100 ml) and extracted using EtOAc
(5.times.150 ml). The combined organic phases were dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
oily residue was purified by sublimation in a high vacuum (p=0.1
torr, oil bath temperature 1100.degree. C.).
2-(4-chloro-2-nitrophenyl)ethanol (13.23 g, 55%) was obtained in
the form of yellow crystals.
R.sub.f (SiO.sub.2, toluene/EtOAc 10:1) 0.26
melting point: 61.degree. to 64.degree. C.
UV(MeOH), .lambda..sub.max [nm] (log 68 ): 213 (4.29), 252 (3.61),
294 (shoulder, 3.10)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.94 (d, J=2.2, H--C(3)); 7.53
(dd, H--C(5)); 7.40 (d, H--C(6)); 3.93 (t, .alpha.-CH.sub.2); 3.14
(t, .beta.-CH.sub.2); 1.70 (s, OH)
Anal. calcd. for C.sub.8 H.sub.8 ClNO.sub.3 (201.61): C 47.66, H
4.00, N 6.95; found: C 47.69, H 4.01, N 6.76
Literature
[1] J. Bakke, Acta Chem. Scand. 1969, 23, 3055
[2] T. Morimoto, I. Hashimoto, H. Yamaoka, Chem. Abstr. 1978, 88,
104880 v
[3] Y. Tsuji, S. Kotachi, K.-T. Huh, Y. Watanabe, J. Org. Chem.
1990, 55, 580
b) 2-(4-chloro-2-nitrophenyl)ethoxycarbonyl chloride
Phosgene was introduced in a solution of
2-(4-chloro-2-nitrophenyl)ethanol (6.8 g, 34 mmol) in THF (50 ml,
dist. over CaH.sub.2) at room temperature. After 2.5 h, the excess
phosgene and the solvent were removed by distillation in a high
vacuum. 2-(4-chloro-2-nitrophenyl)ethoxycarbonyl chloride (8.53 g,
95%) was obtained as a yellow oil.
R.sub.f (SiO.sub.2, CHCl.sub.3) 0.85
UV(CH.sub.3 CN), .lambda..sub.max [nm] (log .epsilon.): 213 (4.26);
254 (3.63); 300 (shoulder, 3.14)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.03 (d, H--C(3)); 7.59 (dd,
H--C(5)); 7.36 (d, H--C(6)); 4.62 (t, .alpha.-CH.sub.2); 3.32
(.beta.-CH.sub.2)
Anal. calcd. for C.sub.9 H.sub.7 Cl.sub.2 NO.sub.4 (264.06): C
40.94, H 2.67, N 5.30; found: C 40.97, H 2.69, N 5.00
c) 5'-O-(2-(4-chloro-2-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (150 mg, 0.62 mmol) was co-evaporated with pyridine
(2.times.5 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine and cooled to
-30.degree. C. A solution of
2-(4-chloro-2-nitrophenyl)ethoxycarbonyl chloride (255 mg, 0.97
mmol) in CH.sub.2 Cl.sub.2 (3 ml, dist. over CaH.sub.2) was added
dropwise thereto for 5 min. After being stirred for 4 h in
conditions of i-PrOH/N.sub.2 cooling (-30.degree. to -15.degree.
C.), the mixture was diluted with CH.sub.2 Cl.sub.2 (30 ml) and
washed with H.sub.2 O (30 ml). The aqueous phase was post-extracted
with CH.sub.2 Cl.sub.2 (2.times.30 ml). The combined organic phases
were dried over MgSO.sub.4, filtered and co-evaporated with toluene
(4.times.10 ml) and CH.sub.2 Cl.sub.2 (20 ml). The crude product
was purified by column chromatography (SiO.sub.2 15.times.3 cm,
solvent: CH.sub.2 Cl.sub.2 /MeOH 100:7 700 ml).
5'-O-(2-(4-chloro-2-nitrophenyl)ethoxycarbonyl)thymidine (219 mg,
75% ) was obtained as a colorless solid.
R.sub.f (SiO2, toluene/EtOAc/MeOH 5:4:1) 0.36
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 212 (4.43), 261
(4.08), 310 (shoulder, 3.07)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.75 (s (br), NH); 7.94 (d,
H--C(3) of CNPEOC); 7.52 (dd, H--C(5)); 7.31 (m, H--C(6) of CNPEOC,
H--C(6) of thymine); 6.32 (t, H--C(1')); 4.41 (m, H--C(3'),
2.times.H--C(5'), .alpha.-CH.sub.2 of CNPEOC); 4.12 (q, H--C(4'));
3.27 (t, .beta.-CH.sub.2 of CNPEOC); 2.75 (s, (br), OH--C(3'));
2.39 (m, H--C(2')), 2.19 (m, H--C (2')); 1.86 (s, CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.20 ClN.sub.3 O.sub.9 (469.83): C
48.57, H 4.29, N 8.94; found: C 48.87, H 4.56, N 8.64
EXAMPLE 7
a) 2-(5-methoxy-2-nitrophenyl)ethanol [1, 2]
Triton B (2 ml, 35% in MeOH) was added at 80.degree. C. to a
mixture of 5-methoxy-2-nitrotoluene (25 g, 150 mmol) and
paraformaldehyde (2.3 g, 73 mmol) in DMSO (20 ml, synthesis
quality, additionally dried for 2 d over molecular sieve 4 .ANG.).
After being stirred 2.5 h at this temperature, it was cooled to
room temperature and neutralized with a few drops of conc. HCl. The
mixture was diluted with H.sub.2 O (50 ml) and extracted with EtOAc
(5.times.100 ml). The organic phases were dried over MgSO.sub.4,
filtered and concentrated under reduced pressure. The residue was
subjected to a distillation in a high vacuum (p=0.1 torr), whereby
a fraction containing starting material (12.1 g) was obtained at
70.degree. C. The residual crude product was purified by column
chromatography (240 g SiO.sub.2, solvent: toluene/EtOAc 8:1 1400
ml, 7:1 1600 ml, 6:1 400 ml, 5:1 400 ml, 3:1 400 ml, 2:1 600 ml and
1:1 600 ml). 2-(5-methoxy-2-nitrophenyl)ethanol (6.87 g, 48%) was
obtained as a yellow oil.
R.sub.f (SiO.sub.2, toluene/EtOAc 1:1) 0.43
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 204 (4.00), 231
(3.82); 301 (3.84)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.04 (dd, H--C(4); 6.82 (m,
H--C(3), H--C(6)); 3.94 (q, .alpha.-CH.sub.2); 3.87 (s, OCH.sub.3);
3.20 (t, .beta.-CH.sub.2); 1.64 (s (br), OH)
Anal. calcd. for C.sub.9 H.sub.11 NO.sub.4 (197.19): C 54.82, H
5.62, N 7.10; found: C 54.78, H 5.86, N 7.00
Literature
[1] T. Morimoto, I. Hashimoto, H. Yamaoka, Chem. Abstr. 1978, 88,
104880 v
[2] Y. Tsuji, S. Kotachi, K.-T. Huh, Y. Watanabe, J. Org. Chem.
1990, 55, 580
b) 2-(5-methoxy-2-nitrophenyl)ethoxycarbonyl chloride
Phosgene was introduced in a solution of
2-(5-methoxy-2-nitrophenyl)ethanol (3.0 g, 15 mmol) in THF (40 ml,
dist. over CaH.sub.2) at room temperature under stirring. After 2.5
h the excess phosgene and the solvent were removed by distillation
in a high vacuum. 2-(5-methoxy-2-nitrophenyl)ethoxycarbonyl
chloride (3.72 g, 96%) was obtained as a yellow oil.
R.sub.f (SiO2, CHCl.sub.3) 0.79
UV(CH.sub.3 CN), .lambda..sub.max [nm] (log .epsilon.): 222
(shoulder, 3.87), 230 (3.90), 303 (3.87)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.12 (d, H--C(3)); 6.88 (dd,
H--C (4)); 6.79 (d, J=2.8, H--C(6)), 4.63 (t, .alpha.-CH.sub.2);
3.89 (s, OCH.sub.3); 3.35 (t, .beta.-CH.sub.2)
Anal. calcd. for C.sub.10 H.sub.10 ClNO.sub.5 : C 46.26, H 3.88, N
5.39; found: C 46.42, H 4.00, N 5.50
c) 5'-O-(2-(5-methoxy-2-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (150 mg, 0.62 mmol) was co-evaporated with pyridine
(2.times.5 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (5 ml, see above)
and cooled to -30.degree. C. (i-PrOH/N.sub.2). A solution of
2-(5-methoxy-2-nitrophenyl)ethoxycarbonyl chloride (250 mg, 0.96
mmol) in CH.sub.2 Cl.sub.2 (5 ml, dist. over CaH.sub.2) was added
dropwise thereto for 5 min. After being stirred for a total of 4 h
in conditions of i-PrOH/N.sub.2 cooling (-30.degree. to -15.degree.
C.), the mixture was diluted with CH.sub.2 Cl.sub.2 (30 ml) and
washed with H.sub.2 O (30 ml). The aqueous phase was post-extracted
with CH.sub.2 Cl.sub.2 (2.times.30 ml). Drying of the organic
phases over Na.sub.2 SO.sub.4, filtering, evaporating the solvent
under reduced pressure and co-evaporating with toluene (4.times.10
ml) and CH.sub.2 Cl.sub.2 (10 ml) yielded the crude product which
was purified by column chromatography (SiO.sub.2, 16.times.3 cm,
CH.sub.2 Cl.sub.2 /MeOH 100:6 800 ml).
5.dbd.-O-(2-(5-methoxy-2-nitrophenyl)ethoxycarbonyl)thymidine (205
mg, 71%) was obtained as a colorless solid.
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.32
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.30), 234
(shoulder, 3.94), 270 (4.09), 302 (shoulder, 3.84)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.79 (s, NH); 8.06 (H--C(3) of
MNPEOC); 7.34 (s, H--C(6) of thymine); 6.85 (dd, H--C(4) of
MNPEOC); 6.77 (d, J=2.7, H--C(6) of MNPEOC); 6.32 (t, H--C(1'));
4.43 (m, H--C(3'), 2.times.H--C(5'), .alpha.-CH.sub.2 of MNPEOC);
4.12 (q, H--C(4')); 3.85 (s, OCH.sub.3); 3.33 (m, .beta.-CH.sub.2
of MNPEOC); 2.75 (d, OH--C(3')); 2.38 (m, H--C(2')); 2.18 (m,
H--C(2')); 1.83 (s, CH.sub.3)
Anal. calcd. for C.sub.20 H.sub.23 N.sub.3 O.sub.10 (465.42): C
51.61, H 4.98, N 9.03; found: C 51.31, H 5.09, N 8.63
EXAMPLE 8
a) 2-(2,4-dichloro-6-nitrophenyl)ethanol
A solution of potassium tertiary-butylate (90 mg, 0.8 mmol) in
tert.-butanol (1 ml, synthesis quality, 99%) was added to a mixture
of 2,4-dichloro-6-nitrotoluene (1.03 g, 5 mmol) and
paraformaldehyde (150 mg, 5 mmol) in DMSO (2.5 ml, synthesis
quality, additionally dried for 2 d over molecular sieve 4 .ANG.).
After the addition of the potassium tertiary-butylate solution, a
color change from yellow to deep violet occurred. It was stirred
for 5 min at room temperature and for 30 min at 70.degree. to
80.degree. C. (oil bath temperature). The mixture was then allowed
to cool to room temperature and neutralized with a few drops of
conc. HCl. The mixture was diluted with EtOAc (30 ml) and washed
with H.sub.2 O (30 ml). The aqueous phase was post-extracted with
EtOAc (2.times.30 ml). Drying the organic phases over Na.sub.2
SO.sub.4, filtering and evaporating the solvent under reduced
pressure yielded the crude product which was purified by column
chromatography (20 g SiO.sub.2, 12.times.2 cm, solvent: PE 30 ml,
PE/EtOAc 10:1 110 ml, 9:1 100 ml, 8:1 360 ml, 7:1 80 ml).
2-(2,4-dichloro-6-nitrophenyl)ethanol (769 mg, 65%) was obtained as
a yellow solid.
R.sub.f (SiO.sub.2, toluene/EtOAc 9:1) 0.41
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.32), 218
(4.20), 254 (shoulder, 3.40), 292 (3.08)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.73 (d, J=2.0, 1 arom H); 7.65
(d, J=2.0, 1 arom. H); 3.92 (m, .alpha.-CH.sub.2); 3.26 (t,
.beta.-CH.sub.2); 1.70 (s (br), OH)
Anal. calcd. for C.sub.8 H.sub.7 Cl.sub.2 NO.sub.3 (236.054): C
40.71, H 2.99, N 5.93; found: C 40.36, H 2.96, N 5.85
b) 2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl chloride
For 5 min, a solution of 2-(2,4-dichloro-6-nitrophenyl)ethanol (500
mg, 2.12 mmol) and Et.sub.3 N (214 mg, 2.12 mmol, dist. over KOH)
in THF (5 ml, dist. over CaH.sub.2) was dropped into a solution,
cooled to 0.degree. C., of trichloromethyl chloroformate (503 mg,
2.5 mmol) in THF (5 ml, see above). It was stirred for 1 h while
being cooled in an ice bath and stirred for 2 h at room
temperature. The mixture was then filtered over Celite. Rewashing
the filter cake with THF and removing the solvent and excess
reagent from the pooled filtrates by distillation at 30.degree. C.
in a high vacuum yielded
2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl chloride (597 mg, 94%)
as a bright yellow solid.
R.sub.f (SiO2, PE/EtOAc 19:1) 0.57
UV(MeOH) .lambda..sub.max [nm] (log .epsilon.): 204 (4.35), 217
(4.25), 252 (shoulder, 3.47), 295 (3.10)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.82 (d, J=1.8, arom. H); 7.70
(d, J=1.8, arom. H); 4.59 (t, .alpha.-CH.sub.2); 3.42 (t,
.beta.-CH.sub.2)
Anal. calcd. for C.sub.9 H.sub.6 Cl.sub.3 NO.sub.4 (298.509): C
36.21, H 2.03, N 4.69; found: C 36.37, H 2.30, N 4.60
c)
51'-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (200 mg, 0.83 mmol) was co-evaporated with pyridine
(3.times.3 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (3 ml, see above)
and cooled to -60.degree. C. (i-PrOH/N.sub.2). A solution of
2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl chloride (320 mg, 1.07
mmol) in CH.sub.2 Cl.sub.2 (3 ml, dist. over CaH.sub.2) was added
dropwise for 15 min. After being stirred for a total of 6 hours in
conditions of i-PrOH/N.sub.2 cooling (-60.degree. to -15.degree.
C.), the mixture was diluted with CH.sub.2 Cl.sub.2 (10 ml) and
washed with H.sub.2 O (10 ml). The aqueous phase was post-extracted
with CH.sub.2 Cl.sub.2 (2.times.10 ml). Drying the organic phases
over Na.sub.2 SO.sub.4, filtering, evaporating the solvent under
reduced pressure and co-evaporating with toluene (4.times.10 ml)
yielded the crude product (543 mg) which was purified by column
chromatography (20 g SiO.sub.2, 12.5.times.2.1 cm, solvent:
CH.sub.2 Cl.sub.2 /MeOH 100:1 50 ml, 100:2 204 ml, 100:3 360 ml).
First
3',5'-bis-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine,
then 3'-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine
and 5'-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine
were eluted. The product fractions were concentrated under reduced
pressure and dried in a high vacuum.
3',5'-bis-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine
(53 mg, 8%) was obtained as a pale yellow foam, while
3'-O-(2(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine (14 mg,
3% ) and
5'-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine (339
mg, 81% ) were each obtained as a colourless foam.
5'-O-(2-(2,4-dichloro-6-nitrophenyl)ethoxycarbonyl)thymidine
R?.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.40
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 203 (4.51), 214
(shoulder, 4.38), 263 (4.02), 304 (shoulder, 3.06)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.91 (s, NH); 7.75 (d, J=2.1, 1
arom. H of DCNPEOC); 7.68 (d, J=2.1, 1 arom. H of DCNPEOC); 7.37
(s, H--C(6) of thymine); 6.37 (t, H--C(1')); 4.63 (m, H--C(3'),
2.times.H--C(5'), .alpha.-CH.sub.2 of DClNPEOC); 4.16 (m,
H--C(4')); 3.40 (t, .beta.-CH.sub.2 of DClNPEOC); 2.86 (d,
OH--C(3')); 2.42 (m, H--C(2')); 2.24 (m, H--C(2')); 1.89 (s,
CH.sub.3)
Anal. calcd. for C.sub.19 H.sub.19 Cl.sub.2 N.sub.3 O.sub.9
(504.279): C 45.25, H 3.80, N 8.33, found: C 45.02, H 3.89, N
8.04
EXAMPLE 9
a) 2-(4,5-dimethoxy-2-nitrophenyl)ethanol [1]
For 6 min, conc. HNO.sub.3 (4.8 ml, 65%, d=1.4) was added dropwise
to a mixture of homoveratryl alcohol (3.02 g, 16.6 mmol) in glacial
acetic acid (30 ml) under stirring, this mixture having been cooled
to -10.degree. C. (common salt/ice) It was then allowed to warm up
to 23.degree. C. in 30 min. After being stirred for 1 h at this
temperature, the mixture was diluted with H.sub.2 O, neutralized
with NaHCO.sub.3 and extracted with EtOAc (3.times.30 ml). The
combined organic phases were dried over Na.sub.2 SO.sub.4, filtered
and concentrated under reduced pressure. The residue (3.25 g) was
purified by column chromatography (90 g SiO.sub.2 toluene/EtOAc 4:1
500 ml, toluene/EtOAc 3:1 400 ml, toluene/EtOAc 2:1 300 ml).
2-(4,5-dimethoxy-2-nitrophenyl)ethanol (2.13 g, 56%) was obtained
as a yellow solid.
R.sub.f (SiO.sub.2, toluene/EtOAc 2:1) 0.24
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 202 (4.18), 216
(4.06), 242 (3.97), 297 (3.63), 340 (3.70)
.sup.1 H-NMR-Spektrum (250 MHz, CDCl.sub.3): 7.61 (s, H--C(3));
6.80 (s, H--C(6)); 3.97 (s, OCH.sub.3); 3.96 (m, .alpha.-CH.sub.2,
unresolved); 3.95 (s, OCH.sub.3); 3.21 (t, .beta.-CH.sub.2), 1.70
(s (br), OH)
Anal. calcd. for C.sub.10 H.sub.13 NO.sub.5 (227.216): C 52.86, H
5.77, N 6.16; found: C 52.87, H 5.82, N 6.12
Literature
[1] E. McDonald, R. D. Wylie, Tetrahedron 1979, 35, 1415
b) 2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl chloride
Trichloromethyl chloroformate (0.26 ml, 2.2 mmol) in THF (5 ml,
dist. over CaH.sub.2) was cooled by means of an ice bath to
0.degree. C. A solution of 2-(4,5-dimethoxy-2-nitrophenyl)ethanol
(500 mg, 2.2 mmol) and Et.sub.3 N (218 mg, 0.3 ml, 2.2 mmol, dist.
over CaH.sub.2) in THF (15 ml, dist. over CaH.sub.2) was added
dropwise thereto for 10 min. The ice bath was then removed and the
solution was stirred further at room temperature. After being
stirred for 30 min, a spatula tip of activated carbon was added to
the reaction mixture, which after being stirred for another 2.5 h
was filtered by suction at room temperature over Celite. The
solvent and the excess reagent were removed from the filtrate by
distillation in a high vacuum and
2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl chloride (624 mg,
98%) was obtained as a yellow brown solid.
R.sub.f (SiO.sub.2, toluene/EtOAc 2:1) 0.77
UV (MeOH), .lambda..sub.max [nm] (log .epsilon.): 203 (4.18), 217
(4.08), 242 (4.01), 297 (3.67), 339 (3.73)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.67 (s, H--C(3)); 6.74 (S,
H--C(6); 4.66 (t, .alpha.-CH.sub.2); 3.99 (s, OCH.sub.3); 3.96 (s,
OCH.sub.3); 3.36 (t, .beta.-CH.sub.2)
Anal. calcd. for C.sub.11 H.sub.12 NO.sub.6 Cl (289.671): C 45.61,
H 4.18, N 4.84; found: C 45.59, H 4.26, N 4.94
c)
51'-O-(2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (200 mg, 0.83 mmol) was co-evaporated with pyridine
(2.times.2 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (2.4 ml, see above)
and cooled to -60.degree. C. (i-PrOH/N.sub.2). A solution of
2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl chloride (361 mg,
1.25 mmol) in CH.sub.2 Cl.sub.2 (2.4 ml, dist. over CaH.sub.2) was
added dropwise thereto for 15 min. After being stirred for a total
of 6.5 h in conditions of i-PrOH/N.sub.2 cooling (-40.degree. to
-15.degree. C.), the mixture was diluted with CH.sub.2 Cl.sub.2 (10
ml) and washed with H.sub.2 O (10 ml). The aqueous phase was
post-extracted with CH.sub.2 Cl.sub.2 (2.times.10 ml). Drying the
organic phases over Na.sub.2 SO.sub.4, filtering, evaporating the
solvent under reduced pressure and co-evaporating with toluene
(4.times.20 ml) yielded the crude product (530 mg) which was
purified by column chromatography (20 g SiO.sub.2, CH.sub.2
Cl.sub.2 /MeOH 100:3 309 ml, 100:4 104 ml, 100:5 160 ml). First
3',5'-bis-O-(2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl)thymidine
and then
5'-O-(2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl)thymidine were
eluted. The product fractions were concentrated under reduced
pressure and dried in a high vacuum.
3',5'-bis-O-(2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl)thymidine
(154 mg, 25%) and
5'-O-(2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl)thymidine (272
mg, 66%) were each obtained as bright yellow solids.
5'-O-(2-(4,5-dimethoxy-2-nitrophenyl)ethoxycarbonyl)thymidine
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.23
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 203 (4.33), 212
(shoulder, 4.30), 247 (4.13), 267 (4.03), 343 (3.69)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 9.10 (s, NH); 7.62 (s,
H--C(3)); 7.37 (d, J=1.2, H--C(6) of thymine); 6.73 (s, H--C(6));
6.35 (t, H--C(1')); 4.44 (m, H--C(3'), 2.times.H--C(5'),
.alpha.-CH.sub.2 of DMNPEOC); 4.15 (m, H--C(4')); 3.96 (s,
OCH.sub.3); 3.94 (s, OCH.sub.3); 3.33 (dt, .beta.-CH.sub.2 of
DMNPEOC); 3.06 (s (br), OH--C(3')); 2.42 (m, H--C(2')); 2.20 (m,
H--C(2')); 1.88 (d, J=0.9, CH.sub.3)
Anal. calcd. for C.sub.21 H.sub.25 N.sub.3 O.sub.11 (495.441): C
50.91, H 5.09, N 8.48, found: C 50.94, H 5.09, N 8.32
EXAMPLE 10
a) 2-(2-nitrophenyl)propanol [1, 2]
A solution of potassium tert.-butylate (360 mg, 3.2 mmol) in
tert.-butanol (4 ml, dist. over CaH.sub.2) was added to
2-nitroethylbenzene (3.02 g, 20 mmol) and paraformaldehyde (600 mg,
20 mmol) in DMSO (10 ml, synthesis quality, additionally dried for
2 d over molecular sieve 4 .ANG.). It was stirred for 15 min at
room temperature and for 1 h 45 min at 70.degree. C. (oil bath
temperature). It was then allowed to cool to room temperature and
neutralized with a few drops of conc. HCl. The mixture was diluted
with EtOAc (100 ml) and washed with a saturated NaCl solution (60
ml). The aqueous phase was post-extracted with EtOAc (2.times.100
ml). Drying the organic phases over Na.sub.2 SO.sub.4, filtering
and evaporating the solvent under reduced pressure yielded the
crude product (5.06 g) which was purified by column chromatography
(80 g SiO.sub.2, 19.times.3.4 cm, solvent: toluene 150 ml,
toluene/EtOAc 8:1 270 ml, 7:1 240 ml, 6:1 280 ml, 5:1 180 ml).
2-(2-nitrophenyl)propanol (2.539 g, 70%) was obtained as a bright
yellow oil.
R.sub.f (SiO.sub.2, toluene/EtOAc 9:1) 0.25
UV (MeOH), .lambda..sub.max [nm] (log .epsilon.): 206 (4.08), 220
(shoulder, 3.75), 254 (3.53), 285 (shoulder, 3.27)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.75 (dd, J=1.2, 8.1, 1 arom.
H, ortho to NO.sub.2); 7.55 (m, 2 arom. H); 7.36 (m, 1 arom. H);
3.80 (m, .alpha.-CH.sub.2); 3.52 (sextett, .beta.-CH); 1.67 (s
(br), OH); 1.33 (d, J=6.9, CH.sub.3)
Anal. calcd. for C.sub.9 H.sub.11 NO.sub.3 (181.191): C 59.66, H
6.12, N 7.73; found: C 59.55, H 6.12, N 7.90
Literature
[1] J. Org. Chem. 1986, 3143
[2] Chem. Abstr. 1989, 110, P 75032 k.
b) 2-(2-nitrophenyl)propoxycarbonyl chloride
A solution of 2-(2-nitrophenyl)propanol (500 mg, 2.76 mmol) and
Et.sub.3 N (279 mg, 0.385 ml, 2.76 mmol, dist. over KOH) in THF
(6.75 ml, dist. over CaH.sub.2) was dropped into a solution, cooled
to 0.degree. C., of trichloromethyl chloroformate (655 mg, 3.3
mmol) in THF (6.75 ml, see above). It was stirred for 1 h while
being cooled in an ice bath and stirred for 1 h at room
temperature. The mixture was filtered over Celite. Rewashing the
filter cake with THF and removing the solvent and excess reagent
from the pooled filtrates by distillation at 30.degree. C. in a
high vacuum yielded 2-(2-nitrophenyl)propoxycarbonyl chloride (644
mg, 96%) as a light brown oil.
R.sub.f (SiO.sub.2, PE/EtOAc 19:1) 0.24
UV (MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.07); 218
(shoulder, 3.75), 251 (3.59)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.84 (dd, J=1.2, 8.0, 1 arom.
H, ortho to NO.sub.2); 7.62 (m, 1 arom. H); 7.44 (m, 2 arom. H);
4.50 (d, J=6.3, .alpha.-CH.sub.2); 3.80 (sextett .beta.-CH.sub.2);
1.42 (d, J=7.0, CH.sub.3)
Anal. calcd. for C.sub.10 H.sub.10 ClNO.sub.4 (243.646): C 49.30, H
4.14, N 5.75; found: C 49.71, H 4.32, N 5.70
c) 5'-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine
Thymidine (1 g, 4.1 mmol) was co-evaporated with pyridine
(3.times.15 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (15 ml, see above)
and cooled to -60.degree. C. (i-PrOH/N.sub.2). A solution of
2-(2-nitrophenyl)propoxycarbonyl chloride (1.31 g, 5.38 mmol) in
CH.sub.2 Cl.sub.2 (18 ml, dist. over CaH.sub.2) was added dropwise
thereto for 30 min. It was stirred for another 6 h in conditions of
i-PrOH/N.sub.2 cooling (-60.degree. to -20.degree. C.). The
reaction mixture was diluted with CH.sub.2 Cl.sub.2 (50 ml) and
washed with H.sub.2 O (50 ml). The aqueous phase was post-extracted
with CH.sub.2 Cl.sub.2 (3.times.50 ml). Drying the organic phases
over Na.sub.2 SO.sub.4, filtering, evaporating the solvent under
reduced pressure and co-evaporating with toluene (4.times.15 ml)
yielded the crude product (2.22 g) which was purified by column
chromatography (80 g SiO.sub.2, 19.times.3.4 cm, solvent: CH.sub.2
Cl.sub.2 /MeOH 100:1 101 ml, 100:2 204 ml, 100:3 206 ml, 100:3.5
207 ml, 100:4 520 ml, 100:5 53 ml, 100:6 53 ml). First
3',5'-bis-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine, then
3'-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine and finally
5'-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine were eluted. After
the product fractions were concentrated under reduced pressure and
dried in a high vacuum,
3',5'-bis-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine (145 mg,
5%) was obtained as a pale yellow foam, while
3'-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine (71 mg, 4% ) and
5'-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine (1.307 g, 71%)
were each obtained as colorless foams.
5'-O-(2-(2-nitrophenyl)propoxycarbonyl)thymidine
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.43
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 207 (4.30), 263
(4.07)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.66 (s, NH, diastereomer);
8.64 (s, NH, diastereomer); 7.77 (m, 1 arom. H of NPPOC); 7.59 (t,
1 arom. H of NPPOC); 7.43 (m, 2 arom. H of NPPOC); 7.33 (s, H--C(6)
of thymine, diastereomer); 7.30 (s, H--C(6) of thymine,
diastereomer), 6.34 (t, H--C(1'), diastereomer); 6.32 (t, H--C(1'),
diastereomer); 4.29 (m, H--C(3'), H--C(4'), 2.times.H--C(5'),
.alpha.-CH.sub.2 of NPPOC); 3.80 (m, .beta.-CH of NPPOC); 2.62 (d,
J=4.2, OH--C(3'), diastereomer); 2.60 (d, J=4.4, OH--C(3'),
diastereomer); 2.39 (m, H--C(2')); 2.18 (m, H--C(2')); 1.86 (s,
CH.sub.3 of thymine, diastereomer); 1.75 (s, CH.sub.3 of thymine,
diastereomer); 1.38 (d, J=7.0, CH.sub.3 of NPPOC, diastereomer);
1.37 (d, J=7.0, CH.sub.3 of NPPOC, diastereomer)
Anal. calcd. for C.sub.20 H.sub.23 N.sub.3 O.sub.9 (449.416): C
53.45, H 5.16, N 9.35; found: C 53.14, H 5.21, N 9.16
EXAMPLE 11
a) 2-chloro-2-(2-nitrophenyl)ethanol
A solution of 2-nitrobenzyl chloride (4.3 g, 25 mmol) in DMSO (3.5
ml, synthesis quality, additionally dried for 2 d over molecular
sieve 4 .ANG.) was mixed with paraformaldehyde (750 mg, 25 mmol)
and DBU (0.5 ml, 3.3 mmol) and stirred at room temperature for 20
min. The pH value of the reaction mixture was adjusted to about pH
3 by means of a few drops of conc. AcOH. The mixture was diluted
with CH.sub.2 Cl.sub.2 (120 ml), washed with H.sub.2 O (80 ml) and
post-extracted with CH.sub.2 Cl.sub.2 (2.times.100 ml).
The organic phases were dried over Na.sub.2 SO.sub.4, filtered and
concentrated under reduced pressure. The crude product (5.543 g)
was purified by column chromatography (150 g SiO.sub.2,
22.times.4.1 cm, solvent: toluene 150 ml, toluene/EtOAc 25:1 260
ml, 15:1 320 ml, 10:1 330 ml, 5:1 720 ml, 4:1 250 ml). First the
starting material (2.587 g, 60%), then a mixed fraction (267 mg)
and finally 2-chloro-2-(2-nitrophenyl)ethanol (1.178 g, 23% and 61%
in relation to the consumed starting material) were eluted. The
mixed fraction obtained (267 mg) was purified by a further column
chromatography (8 g SiO.sub.2, 15.times.1.2 cm; solvent: toluene 50
ml, toluene/EtOAc 100:1 50 ml). First a further starting material
(44 mg, 1%) and then 2-nitrostyrene epoxide (68 mg, 2% and 4% in
relation to the consumed starting material) were eluted. The total
amount of re-isolated starting material was 2.631 g (61%).
2-chloro-2-(2-nitrophenyl)ethanol (yellow solid)
R.sub.f (SiO.sub.2, CH.sub.2 Cl.sub.2) 0.23
melting point: 49.degree. to 50.degree. C.
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 209 (4.11), 254
(3.64), 332 (shoulder, 2.70)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 7.94 (dd, J=1.3, 8.3, 1 arom.
H); 7.89 (dd, J=1.3, 7.9, 1 arom. H); 7.69 (m, 1 arom. H); 7.51 (m,
1 arom. H); 5.73 (dd, J=4.6, 6.8, .beta.-CH); 4.11 (dd, J=4.6,
12.1, .alpha.-CH); 4.00 (dd, J=6.8, 12.0, .alpha.-CH); 2.14 (s,
OH)
.sup.1 H-NMR (250 MHz, DMSO-d.sub.6): 7.94 (dd, J=1.2, 8.1, 1 arom.
H); 7.86 (dd, J=1.5, 7.9, 1 arom. H); 7.77 (dt, J=1.2, 7.4, 1 arom.
H); 7.60 (dt, J=1.6, 8.3, 1 arom. H); 5.43 (t, J=6.4, .beta.-CH; s
(br), partially concealed, OH), 3.86 (d, J=6.4,
.alpha.-CH.sub.2)
Anal. calcd. for C.sub.8 H.sub.8 ClNO.sub.3 (201.609): C 47.66, H
4.00, N 6.95; found: C 48.01, H 4.11, N 7.00
b) 2-chloro-2-(2-nitrophenyl)ethoxycarbonyl chloride
A solution of diphosgene (1.7 ml, 14 mmol) in THF (10 ml, dist.
over CaH.sub.2) was cooled by means of an ice bath to about
0.degree. C. A solution of 2-chloro-2-(2-nitrophenyl)ethanol (705
mg, 3.5 mmol) and Et.sub.3 N (354 mg, 0.485 ml, 3.5 mmol) in THF
(10 ml, dist. over CaH.sub.2) was dropped thereto for about 30 min.
After the solution had been stirred for a further hour, the ice
bath was removed. The mixture was stirred for another 3 h at room
temperature. The mixture was filtered over Celite. The precipitate
was rewashed with THF (10 ml, dist. over CaH.sub.2). The combined
filtrates were concentrated under reduced pressure and then dried
in a high vacuum at room temperature.
2-chloro-2-(2-nitrophenyl)ethoxycarbonyl chloride (896 mg, 97%) was
obtained as a light brown oil.
R.sub.f (SiO.sub.2, CH.sub.2 Cl.sub.2) 0.82
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 208 (4.11); 253
(3.66)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.01 (dd, J=1.1, 8.1, 1 arom.
H); 7.93 (dd, J=1.1, 7.9, 1 arom. H); 7.74 (dt, J=1.1, 8.2, 1 arom.
H); 7.57 (m, 1 arom. H); 5.89 (dd, J=4.9, 6.4, .beta.-CH); 4.80
(dd, J=6.5, 11.4, .alpha.-CH); 4.73 (dd, J=4.9, 11.5,
.alpha.-CH)
Anal. calcd. for C.sub.9 H.sub.7 Cl.sub.2 NO.sub.4 (264.064): C
40.94, H 2.67, N 5.30; found: C 41.39, H 2.89, N 5.38
c) 5'-O-(2-chloro-2-(2-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (250 mg, 1.03 mmol) was co-evaporated with pyridine
(2.times.3 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (3.5 ml, see above)
and cooled to approx. -40.degree. C. (i-PrOH/N.sub.2). A solution
of 2-chloro-2-(2-nitrophenyl) ethoxycarbonyl chloride (356 mg, 1.35
mmol) in CH.sub.2 Cl.sub.2 (3.5 ml, dist. over CaH.sub.2) was
dropped thereto. It was stirred for another 4 h 45 min in
conditions of i-PrOH/N.sub.2 cooling (-40.degree. to -10.degree.
C.). The reaction mixture was diluted with H.sub.2 O (10 ml) and
extracted with CH.sub.2 Cl.sub.2 (4.times.10 ml). The organic
phases were dried over Na.sub.2 SO.sub.4, filtered off,
concentrated under reduced pressure and co-evaporated with toluene
(4.times.10 ml). The crude product (665 mg) was purified by column
chromatography (20 g SiO.sub.2, 12.times.2.1 cm, solvent: CH.sub.2
Cl.sub.2 100 ml, CH.sub.2 Cl.sub.2 /MeOH 100:1 101 ml, 100:2 102
ml, 100:2.5 102 ml, 100:3 206 ml, 1004 104 ml, 100:5 105 ml). First
bis-(2-chloro-2-(2-nitrophenyl)ethyl)carbonate (60 mg, 10%) was
obtained as a yellow oil, then
3',5'-bis-O-(2-chloro-2-(2-nitrophenyl)ethoxycarbonyl)thymidine (48
mg) was obtained as a bright yellow foam, then
3'-O-(2-chloro-2-(2-nitrophenyl)ethoxycarbonyl)thymidine (16 mg,
3%) was obtained a colorless foam and finally
5'-O-(2-chloro-2-(2-nitrophenyl)ethoxycarbonyl)thymidine (394 mg,
81%) as a colorless foam.
5'-O-(2-chloro-2-(2-nitrophenyl)ethoxycarbonyl)thymidine
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.34
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 209 (4.34), 262
(4.10)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.37 (s, (br), NH); 7.97 (d,
J=8.1, 1 arom. H of CNPEOC); 7.91 (dd, J=1.0, 7.9, 1 arom. H of
CNPEOC); 7.72 (t, J=7.3, 1 arom. H of CNPEOC); 7.55 (m, 1 arom. H
of CNPEOC); 7.34 (m, H--C(6) of thymine), 6.35 (dd, J=4.0, 6.7,
H--C(1'), diastereomer); 6.32 (dd, J=3.9, 6.5, H--C(1'),
diastereomer); 5.89 (m, .beta.-CH of CNPEOC); 4.57 (m,
.alpha.-CH.sub.2 of CNPEOC), H--C(3'), 2.times.H--C(5')); 4.15 (q,
J=3.2, H--C(4')); 2.41 (m, H--C(2')); 2.22 (m, H--C(2')); 1.90 (s,
CH.sub.3 of thymine, diastereomer); 1.86 (s, CH.sub.3 of thymine,
diastereomer); 1.61 (s (br), OH--C(3'))
Anal. calcd. for C.sub.19 H.sub.20 ClN.sub.3 O.sub.9 (469.834): C
48.57, H 4.29, N 8.94; found: C 48.17, H 4.40, N 8.47
EXAMPLE 12
2-methoxy-2-(2-nitrophenyl)ethanol
Paraformaldehyde (750 mg, 25 mmol) and DBU (1.85 ml, 12.4 mmol)
were added to a solution of 2-nitrobenzylmethylether (4.18 g, 25
mmol) in DMSO (3.5 ml, synthesis quality, additionally dried for 2
d over molecular sieve 4 .ANG.) and allowed to react at room
temperature for 4 h. The pH value was adjusted from pH 9 to about
pH 3.5 by means of a few drops of conc. AcOH. The mixture was
diluted with CH.sub.2 Cl.sub.2 (120 ml) and washed with H.sub.2 O
(80 ml). The aqueous phase was post-extracted with CH.sub.2
Cl.sub.2 (2.times.80 ml). The combined organic phases were dried
over Na.sub.2 SO.sub.4, filtered and concentrated under reduced
pressure. The crude product (5.56 g) was purified by column
chromatography (140 g SiO.sub.2, 21.times.4.2 cm, solvent: toluene
300 ml, toluene/EtOAc 20:1 210 ml, 15:1 160 ml, 10:1 220 ml, 15:2
170 ml, 6:1 210 ml, 5:1 180 ml, 4:1 400 ml, 3:1 240 ml, 2:1 240
ml). First 2-nitrobenzylmethylether (2.442 g, 58%) was eluted then
2-methoxy-2-(2-nitrophenyl)ethanol (1.696 g, 34% and 82%
respectively, in relation to the consumed starting material).
2-methoxy-2-(2-nitrophenyl)ethanol (yellow solid)
R.sub.f (SiO.sub.2, CH.sub.2 Cl.sub.2 /MeOH 100:3) 0.31
Melting point: 61.degree. to 630.degree. C.
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 206 (4.09), 256
(3.66)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.00 (m, 1 arom. H); 7.70 (m, 2
arom. H); 7.48 (m, 1 arom. H); 4.95 (dd, J=3.2, 7.2, .beta.-CH);
3.94 (ddd, J=3.2, 8.8, 11.8, .alpha.-CH); 3.67 (ddd, J=4.4, 7.2,
11.7, .alpha.-CH); 3.31 (s, OCH.sub.3); 2.29 (dd, J=4.4, 8.8,
OH)
Anal. calcd. for C.sub.9 H.sub.11 NO.sub.4 (197.19): C 54.82, H
5.62, N 7.10; found: C 55.14, H 5.57, N 7.15
b) 2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl chloride
A solution of 2-methoxy-2-(2-nitrophenyl)ethanol (986 mg, 5 mmol)
and Et.sub.3 N (506 mg, 0.693 ml, 5 mmol) in THF (10 ml, dist. over
CaH.sub.2) was dropped in a solution, cooled to 0.degree. C., of
disphosgene (1.2 ml 10 mmol) in THF (15 ml, dist. over CaH.sub.2)
for 1 h. After the mixture had been stirred for another 15 min at
0.degree. C., the ice bath was removed and the mixture was stirred
for another 1 h 30 min at room temperature. The mixture was
filtered over Celite and the precipitate rewashed with THF (30 ml,
dist. over CaH.sub.2). The combined filtrates were concentrated
under reduced pressure and dried in a high vacuum at room
temperature. 2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl chloride
(1.264 g, 97%) was obtained as a light brown oil.
R.sub.f (SiO.sub.2, CH.sub.2 Cl.sub.2) 0.73
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.12), 253
(3.71), 348 (shoulder, 2.74)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.07 (dd, J=1.1, 8.2, 1 arom.
H); 7.81 (dd, J=1.6, 7.8, 1 arom. H); 7.73 (dt, J=1.1, 7.6, 1 arom.
H); 7.54 (m, 1 arom. H); 5.13 (dd, J=3.2, 6.3, .beta.-CH); 4.60
(dd, J=3.2, 11.2, .alpha.-CH); 4.54 (dd, J=6.4, 11.3,
.alpha.-CH)
Anal. calcd. for C.sub.10 H.sub.10 ClNO.sub.5 (259.645): C 46.26, H
3.88, N 5.39; found: C 46.74, H 3.88, N 5.40
c) 5'-O-(2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl)thymidine
Thymidine (250 mg, 1.03 mmol) was co-evaporated with pyridine
(2.times.3 ml, pro analysi quality, additionally dried over
molecular sieve 4 .ANG.), dissolved in pyridine (3.5 ml, see above)
and cooled to approx. -50.degree. C. (i-PrOH/N.sub.2). A solution
of 2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl chloride (348 mg, 1.34
mmol) in CH.sub.2 Cl.sub.2 (3.5 ml, dist. over CaH2) was dropped
thereto for approx. 1.5 h. It was stirred for another 3.5 h in
conditions of i-PrOH/N.sub.2 cooling (-40.degree.to -10.degree.
C.). The reaction mixture was diluted with H.sub.2 O (10 ml) and
extracted with CH.sub.2 Cl.sub.2 (3.times.10 ml). The organic
phases were dried over Na.sub.2 SO.sub.4, filtered off,
concentrated under reduced pressure and co-evaporated with toluene
(4.times.10 ml). The crude product (578 mg) was purified by column
chromatography (20 g SiO.sub.2, 12.times.2.1 cm, solvent: CH.sub.2
Cl.sub.2 100 ml, CH.sub.2 Cl.sub.2 /MeOH 100:1 202 ml, 100:2 102
ml, 100:3 103 ml, 100:4 104 ml, 100:5 105 ml, 100:6 106 ml). First
bis(2-methoxy-.sup.2 -(.sub.2 -nitrophenyl)ethyl)carbonate (88 mg,
16%) was obtained as a pale yellow solid and then a mixed fraction
of 3'-O-(2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl)thymidine and
5'-O-(2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl)thymidine (323 mg)
was obtained. The mixed fraction was purified by another column
chromatography (7 g SiO.sub.2, 13.times.1.2 cm, solvent: CH.sub.2
Cl.sub.2 30 ml, CH.sub.2 Cl.sub.2 /MeOH 100:1 50 ml, 100:2 51 ml,
100:2.5 51 ml, 100:3 51 ml, 100:4 52 ml, 100:5 52 ml). First
3'-O-(2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl)thymidine (11 mg,
2%) and then
5'-O-(2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl)thymidine (290 mg,
60%) were each obtained as a colorless foam.
5'-O-(2-methoxy-2-(2-nitrophenyl)ethoxycarbonyl)thymidine
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.40
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 207 (4.33), 263
(4.12)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.67 (s (br), NH); 8.03 (d,
J=5.0, 1 arom. H of MNPEOC); 7.78 (m, 1 arom. H of MNPEOC); 7.70
(t, J=7.5, 1 arom. H of MNPEOC); 7.52 (m, 1 arom. H of MNPEOC);
7.43 (m, H--C(6) of thymine); 6.38 (dd, J=3.8, 6.7, H--C(1'),
diastereomer); 6.35 (dd, J=3.7, 6.4, H--C(1'), diastereomer); 5.14
(m, .beta.-CH of MNPEOC); 4.42 (m, .alpha.-CH.sub.2 of MNPEOC,
H--C(3'), 2.times.H--C(5')); 4.15 (q, J=3.2, H--C(4')); 3.28 (s,
OCH.sub.3, diastereomer); 3.27 (s, OCH.sub.3, diastereomer); 2.64
(s (br), OH--C(3')); 2.42 (m, H--C(2')); 2.23 (m, H--C(2')); 1.94
(s, CH.sub.3 of thymine, diastereomer); 1.92 (s, CH.sub.3 of
thymine, diastereomer)
Anal. calcd. for C.sub.20 H.sub.23 N.sub.3 O.sub.10 (465.415): C
51.51, H 4.98, N 9.03; found: C 51.65, H 5.18, N 8.94
EXAMPLE 13
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.4
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxycytidine
N.sup.4 -(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxycytidine (5 g,
11.9 mmol) was co-evaporated with pyridine (2.times.60 ml, pro
analysi quality, additionally dried over molecular sieve 4 .ANG.),
dissolved in pyridine (60 ml, see above) and cooled to -60.degree.
C. (i-PrOH/N.sub.2). A solution of
2-(2-chloro-6-nitrophenyl)ethoxycarbonyl chloride (4.7 g, 17.8
mmol) in CH.sub.2 Cl.sub.2 (60 ml, dist. over CaH.sub.2) was added
dropwise thereto for 75 min and was stirred for 1 h 45 min in
conditions of i-PrOH/N.sub.2 cooling (-40.degree. to -25.degree.
C.). The mixture was diluted with CH.sub.2 Cl.sub.2 (150 ml) and
washed with H.sub.2 O (100 ml). The aqueous phases were
post-extracted with CH.sub.2 Cl.sub.2 (100 ml). Drying the organic
phases over Na.sub.2 SO.sub.4, filtering, evaporating the solvent
under reduced pressure and co-evaporating with toluene (3.times.50
ml) yielded the crude product (9.8 g) which was purified by column
chromatography (440 g SiO.sub.2, 32.times.5.9 cm, solvent: CH.sub.2
Cl.sub.2 /MeOH 100:4).
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.4
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxycytidine (5.247 g, 680%)
was obtained as a colourless foam.
R.sub.f (SiO.sub.2, CH.sub.2 Cl.sub.2 /MeOH 5:1) 0.82
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (shoulder,
4.56), 211 (4.58), 242 (4.28), 275 (4.17)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.18 (m, 2 arom. H of NPEOC,
ortho to NO.sub.2): 8.04 (d, H--C(6) of Cytosin); 8.03 (s (br), NH,
partially concealed); 7.73 (dd, 1 arom. H of CNPEOC);
7.66 (dd, 1 arom. H of CNPEOC); 7.41 (m, 2 arom. H of NPEOC, meta
to NO.sub.2); 7.37 (t, H--C(4) of CNPEOC, partially concealed);
7.22 (d, H--C(5) of Cytosin), 6.34 (t, H--C(1')); 4.43 (m,
H--C(3'), 2.times.H--C(5'), .alpha.-CH.sub.2 of CNPEOC,
.alpha.-CH.sub.2 of NPEOC); 4.26 (m, H--C(4')); 3.76 (s (br),
OH--C(3')); 3.43 (t, .beta.-CH.sub.2 of CNPEOC); 3.12 (t,
.beta.-CH.sub.2 of NPEOC); 2.73 (m, H--C(2')); 2.14 (m,
H--C(2'))
Anal. calcd. for C.sub.27 H.sub.26 N.sub.5 O.sub.12 Cl (647.981): C
50.05, H 4.04, N 10.81; found: C 49.87, H 4.17, N 10.74
EXAMPLE 14
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.6
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxyadenosine
N.sup.6 -(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxyadenosine (5 g,
11.3 mmol) was co-evaporated with pyridine (2.times.60 ml, pro
analysi quality, additionally dried over molecular sieve 4 .ANG.),
dissolved in pyridine (60 ml, see above) and cooled to -60.degree.
C. (i-PrOH/N.sub.2). A solution of
2-(2-chloro-6-nitrophenyl)ethoxycarbonyl chloride (4.17 g, 15.8
mmol) in CH.sub.2 Cl.sub.2 (60 ml, dist. over CaH.sub.2) was added
dropwise for 2 h. After it had been stirred another 2 h in
conditions of i-PrOH/N.sub.2 cooling (-40.degree. to -25.degree.
C.), the mixture was diluted with CH.sub.2 Cl.sub.2 (150 ml) and
washed with H.sub.2 O (100 ml). The aqueous phases were
post-extracted with CH.sub.2 Cl.sub.2 (100 ml). Drying the organic
phases over Na.sub.2 SO.sub.4, filtering, evaporating the solvent
under reduced pressure and co-evaporating with toluene (3.times.50
ml) yielded the crude product (8.73 g) which was purified by column
chromatography (400 g SiO.sub.2, 28.times.5.7 cm, solvent: CH.sub.2
Cl.sub.2 /MeOH 100:3).
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.6
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxyadenosine (6.318 g, 83%)
was obtained as a colourless foam.
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.33
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 209 (4.67), 266
(4.46)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.71 (S, H--C(8)); 8.53 (s
(br), NH, partially concealed), 8.23 (s, H--C(2)); 8.17 (m, 2 arom.
H of NPEOC, ortho to NO.sub.2); 7.73 (dd, 1 arom. H of CNPEOC);
7.65 (dd, 1 arom. H of CNPEOC); 7.44 (m, 2 arom. H of NPEOC, meta
to NO.sub.2); 7.36 (t, H--C(4) of CNPEOC, partially concealed);
6.54 (t, H--C(1')); 4.77 (m, H--C(3')); 4.55 (t, .alpha.-CH.sub.2
of CNPEOC); 4.41 (m, .alpha.-CH.sub.2 of NPEOC, 2.times.H--C(5'));
4.29 (q, H--C(4')); 3.43 (t, .beta.-CH.sub.2 of CNPEOC); 3.16 (t,
.beta.-CH.sub.2 of NPEOC); 3.15 (s (br), OH--C(3'), partially
concealed); 2.89 (m, H--C(2')); 2.60 (m, H--C(2'))
Anal. calcd. for C.sub.28 H.sub.26 N.sub.7 O.sub.11 Cl (672.007): C
50.05, H 3.90, N 14.59; found: C 49.62, H 3.96, N 14.33
EXAMPLE 15
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl
thymidine-3'-O-((.beta.-cyanoethyl)
(N,N-diisopropylamino)phosphoramidite)
51-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl thymidine (1.9 g, 4
mmol) and 1H-tetrazole (140 mg, 2 mmol) in CH.sub.2 Cl.sub.2 (20
ml, dist. over CaH.sub.2) and CH.sub.3 CN (8 ml, pro analysi
quality) were suspended in a flask under argon;
bis(diisopropylamino) (.beta.-cyanoethoxy)phosphine (1.87 g, 6.2
mmol) was added to this mixture. After it had been stirred for 16.5
h at room temperature, the mixture was diluted with CH.sub.2
Cl.sub.2 (50 ml) and washed with a saturated NaHCO.sub.3 solution
(25 ml). The aqueous phase was post-extracted with CH.sub.2
Cl.sub.2 (2.times.25 ml). Drying the organic phases over Na.sub.2
SO.sub.4, filtering and evaporating the solvent under reduced
pressure yielded the crude product (3.4 g) which was purified by
column chromatography (40 g SiO.sub.2, 12.times.3.1 cm, solvent:
toluene/EtOAc 5:1 160 ml, 4:1 150 ml, 3:1 120 ml, 2:1 150 ml, 1:1
100 ml, 1:2 150 ml, 1:3 160 ml, each with the addition of 1%
Et.sub.3 N by volume).
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl
thymidine-3'-O-((.beta.-cya noethyl)
(N,N-diisopropylamino)phosphoramidite) (2.012 g, 75%) was obtained
as a colorless foam.
R.sub.f (toluene/EtOAc/MeOH 5:4:1) 0.64 and 0.70
(diastereomers)
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.45), 262
(4.06)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.40 (s, NH); 7.74 (td, 1 arom.
H of CNPEOC; 7.66 (dd, 1 arom. H of CNPEOC); 7.38 (m, H--C(4) of
CNPEOC); 6.37 (m, H--C(1')); 4.61 to 4.35 (m, H--C(3'),
2.times.H--C(5'), .alpha.-CH.sub.2 of CNPEOC); 4.28 (m, H--C(4'),
diastereomer); 4.22 (m, H--C(4'), diastereomer); 3.90-3.66 (m,
.alpha.-CH.sub.2 of cyanoethoxy); 3.57 (m, 2.times.NCH); 3.43 (td,
.beta.-CH.sub.2 of CNPEOC); 2.66 (t, .beta.-CH.sub.2 of
cyanoethoxy); 2.48 (m, H--C(2')); 2.23 (m, H--C(2')); 1.86 (d,
CH.sub.3); 1.23 (m, 2.times.NC(CH.sub.3).sub.2)
.sup.31 P-NMR (161.7 MHz, CDCl.sub.3): 149.73 and 149.85 and 149.85
(diastereomers)
Anal. calcd. for C.sub.28 H.sub.37 N.sub.5 O.sub.10 PCl (670.056):
C 50.19, H 5.57, N 10.45; found: C 50.43, H 5.90, N 10.43
EXAMPLE 16
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.4
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxycytidine-3'-O-((.beta.-cyanoeth
yl) (N,N-diisopropylamino)phosphoramidite)
Bis(diisopropylamino) (.beta.-cyanoethoxy)phosphine (1.32 g, 4.38
mmol) was added to
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl-N.sup.4
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxycytidine (1.9 g, 2.9
mmol) and 1H-tetrazole (102 mg, 1.45 mmol) in CH.sub.2 Cl.sub.2 (20
ml, dist. over CaH.sub.2) and CH.sub.3 CN (8 ml, pro analysi
quality) in a flask filled with argon and stirred at room
temperature. After 13.5 h, the solution was diluted with CH.sub.2
Cl.sub.2 (50 ml) and washed with saturated NaHCO.sub.3 solution (25
ml). The aqueous phase was post-extracted with CH.sub.2 Cl.sub.2
(2.times.25 ml). The combined organic phases were dried with
Na.sub.2 SO.sub.4, filtered and concentrated under reduced
pressure. The crude product (2.99 g) was purified by column
chromatography (61 g SiO.sub.2, 18.times.3.2 cm, solvent:
PE/acetone 5:1 170 ml, 4:1 200 ml, 3:1 200 ml, 2:1 600 ml, 3:2 150
ml, 1:1 80 ml, 2:3 300 ml, 1:2 90 ml).
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.4
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxycytidine-3'-O-((.beta.-cyanoeth
yl) (N,N-diisopropylamino)phosphoramidite) (2.29 g, 93%) was
obtained as a colorless foam.
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.61 and 0.67
(diastereomers)
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 203 (4.66), 209
(shoulder, 4.64), 241 (4.30), 276 (shoulder, 4.18)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.18 (m, 2 arom. H of NPEOC),
ortho to NO.sub.2); 8.02 (m, H--C(6) of cytosin); 7.74 (td, 1 arom.
H of CNPEOC); 7.66 (dd, 1 arom. H of CNPEOC); 7.40 (m, 2 arom. H of
NPEOC, meta to NO.sub.2); 7.39 (m, H--C(4) of CNPEOC, partially
concealed); 7.17 (d, H--C(5) of Cytosin); 6.28 (m, H--C(1')); 4.47
to 4.29 (m, .alpha.-CH.sub.2 of NPEOC), .alpha.-CH.sub.2 of
CNPEOC), H--C(3'), H--C(4'), 2.times.H--C(5')); 3.90 to 3.53 (m,
.alpha.-CH.sub.2 of cyanoethoxy, 2.times.NCH); 3.43 (t,
.beta.-CH.sub.2 of CNPEOC); 3.12 (t, .beta.-CH.sub.2 of NPEOC);
2.73 (m, H--C(2') partially concealed); 2.65 (t, .beta.-CH.sub.2 of
cyanoethoxy); 2.17 (m, H--C(2')); 1.23 (m,
2.times.NC(CH.sub.3)2)
.sup.31 P-NMR (161.7 MHz, CDCl.sub.3): 149.67 and 150.02
(diastereomers)
Anal. calcd. for C.sub.36 H.sub.43 N.sub.7 O.sub.13 PCl (848.203):
C 50.98, H 5.11, N 11.56; found: C 50.88, H 5.18, N 11.36
EXAMPLE 17
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.6
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxyadenosine-3'-O-((-.beta.-cyanoe
thyl) (N,N-diisopropylamino)phosphoramidite)
Bis(diisopropylamino) (.beta.-cyanoethoxy)phosphine (1.36 g, 4.51
mmol) was added to a mixture of
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.6
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxyadenosine (2 g, 2.98
mmol) and 1H-tetrazole (104 mg, 1.49 mmol) in CH.sub.2 Cl.sub.2 (20
ml, dist. over CaH.sub.2) and CH.sub.3 CN (8 ml, pro analysi
quality) in a flask filled with argon and stirred for 13.5 h at
room temperature. The solution was diluted with CH.sub.2 Cl.sub.2
(50 ml) and washed with saturated NaHCO.sub.3 solution (25 ml). The
aqueous phase was post-extracted with CH.sub.2 Cl.sub.2 (2.times.25
ml). The combined organic phases were dried with Na.sub.2 SO.sub.4,
filtered and concentrated under reduced pressure. The crude product
was purified by column chromatography (92 g SiO.sub.2, 13.times.4.2
cm, solvent: PE/acetone 6:1 140 ml, 5:1 180 ml, 4:1 200 ml, 3:1 240
ml, 2:1 750, 3:2 150 ml, 1:1 400 ml, 2:3 600 ml, each with the
addition of 1% Et.sub.3 N by volume).
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)-N.sup.6
-(2-(4-nitrophenyl)ethoxycarbonyl)-2'-deoxyadenosine-3'-O-((.beta.-cyanoet
hyl) (N,N-diisopropylamino)phosphoramidite) (2.146 g, 83%) was
obtained as a colorless foam.
R.sub.f (SiO.sub.2, toluene/EtOAc/MeOH 5:4:1) 0.71 and 0.76
(diastereomers)
UV(MeOH), .lambda..sub.max [nm] (log .epsilon.): 205 (4.68), 266
(4.43)
.sup.1 H-NMR (250 MHz, CDCl.sub.3): 8.74 (d, H--C(8) of adenine);
8.23 (s, H--C(2) of adenine); 8.18 (m, 2 arom. H of NPEOC, ortho to
NO.sub.2); 7.73 (m, 1 arom. H of CNPEOC); 7.65 (m, 1 arom. H of
CNPEOC); 7.44 (m, 2 arom. H of NPEOC, meta to NO.sub.2); 7.36 (t,
H--C(4) of CNPEOC); 6.52 (m, H--C(1')); 4.77 (m, H--C(3')); 4.54
(t, .alpha.-CH.sub.2 of CNPEOC); 4.39 (m, .alpha.-CH.sub.2 of
NPEOC, H--C(4'), 2.times.H--C(5')); 3.93 to 3.59 (m, 2.times.NCH,
.alpha.-CH.sub.2 of cyanoethoxy); 3.41 (td, .beta.-CH.sub.2 of
CNPEOC); 3.16 (t, .beta.-CH.sub.2 of NPEOC); 2.90 (m, H--C(2'));
2.71 (m, H--C(2'), partially concealed); 2.67 (m, .beta.-CH.sub.2
of cyanoethoxy); 1.24 (m, 2.times.NC(CH.sub.3).sub.2)
.sup.31 P-NMR (161.7 MHz, CDCl.sub.3): 149.70 and 149.79
(diastereomers)
Anal. calcd. for C.sub.37 H.sub.43 N.sub.9 O.sub.12 PCl (872.229):
C 50.95, H 4.97, N 14.45; found: C 50.92, H 5.11, N 14.21
__________________________________________________________________________
Summary of the Preparation Examples Ex. R.sup.1 R.sup.2 R.sup.3
R.sup.4 R.sup.5 R.sup.6 B
__________________________________________________________________________
1 H H H H H H thymine 2 H H NO.sub.2 H H H thymine 3 H H F H H H
thymine 4 H H Cl H H H thymine 5 H H Br H H H thymine 6 Cl H H H H
H thymine 7 H OCH.sub.3 H H H H thymine 8 Cl H Cl H H H thymine 9
OCH.sub.3 OCH.sub.3 H H H H thymine 10 H H H CH.sub.3 H H thymine
11 H H H Cl H H thymine 12 H H H OCH.sub.3 H H thymine 13 H H Cl H
H H N.sup.4 -(2-pNPEOC)- cytosine 14 H H Cl H H H N.sup.6
-(2-pNPEOC)- adenine 15 H H Cl H ##STR8## H thymine 16 H H Cl H
##STR9## H N.sup.4 -(2-pNPEOC)- cytosine 17 H H Cl H ##STR10## H
N.sup.6 -(2-pNPEOC)- adenine
__________________________________________________________________________
Irradiation experiments
1. Implementation
The corresponding protected thymidine nucleosides were irradiated
by means of an irradiation apparatus which was composed of a Hg
very high pressure lamp (OSRAM HBO, 200 W), a focusing lens, a
shutter with an electronic control device for setting the shutter
times and a temperature-controlled cuvette holder. A heat filter
(0.032 molar CuSO.sub.4 solution) between the lamp and sample was
also installed. 0.2 mM solutions of the nucleosides in MeOH/H.sub.2
O 1:1 were irradiated at 20.degree. to 30.degree. C. with the
entire lamp spectrum (polychromatic light with wavelengths of
.lambda.>289 nm).
The cleavage of the protective groups was followed quantitatively
by HPLC. The HPLC unit comprised of the following devices:
Merck-Hitachi L-6200 Intelligent Pump, Merck-Hitachi Intelligent
Auto Sampler AS 4000, Merck-Hitachi Interface, Merck Lichrosorb
column RP 18, 125.times.5 mm. The ultra-violet/VIS
spectrophotometer was a Kontron UVIKON 730 LC. Detection took place
at 260 nm. The integration of the chromatogram signals took place
by means of Merck software: D-6000 HPLC Manager.
MeOH/H.sub.2 O mixtures were always used for chromatography. The
following gradient was used (flow: 1 ml/min)
______________________________________ time (min) MeOH/H.sub.2 O
1:1 H.sub.2 O MeOH ______________________________________ 0 10 90 0
5 10 90 0 15 90 10 0 30 50 0 50 35 10 90 0
______________________________________
Calibration curves for thymidine and the protected nucleosides were
compiled by injecting dilution series into the chromatograph.
Samples (loop volume: 20 .mu.l) were taken from the irradiated
solutions after specific time intervals. Each sample was injected
twice into the chromatograph. The mean values of these dual
determinations were used for further evaluation. The peak areas of
the chromatograms were converted into the concentrations of
protected nucleosides or of thymidine by means of the
above-compiled calibration curves. The concentration values
obtained in this manner were divided by the maximum achievable
concentration of 0.2 mM (=concentration of the protected
nucleosides at the start of irradiation) and plotted against the
irradiation time as a "relative quantity" in percent. The table
given in section 2.3 shows a result of such a measurement with the
compound 5'-O-(2-(2-chloro-6-nitrophenyl)-ethoxycarbonyl)
thymidine.
2. Examples
2.1 Thymidine calibration
A dilution series with the following concentrations was prepared
for the calibration of thymidine: 0.2 mM, 0.16 mM, 0.12 mM, 0.08 mM
and 0.04 mM. Three injections into the chromatograph were made per
concentration. The calibration line was calculated on the basis of
resultant mean values and the zero point value (0 peak area=0
concentration) by means of linear regression (see table).
______________________________________ Calibration of thymidine
Peak Area Concentration of thymidine (mM)
______________________________________ 516.893 0.20 414.730 0.16
307.575 0.12 208.952 0.08 99.5080 0.04 0.00000 0.00
______________________________________
Linear regression yielded: y=4.96777.multidot.10.sup.-4 +3.85757
10.sup.-7 .multidot.x with x=peak area and y=thymidine
concentration; r=0.9999
2.2 Calibration of
5'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine
A dilution series with the following three concentrations was
prepared: 0.2 mM, 0.14 mM and 0.08 mM. The mean values from three
injections per concentration and the zero point value were used to
calculate the regression line (see table):
______________________________________ Calibration of CNPEOC-T Peak
Area Concentration CNPEOC-T (mM)
______________________________________ 617.057 0.20 435.070 0.14
238.624 0.08 0.00000 0.00
______________________________________
Linear regression yielded: y=9.2999.multidot.10.sup.-4 +3.22516
10.sup.-7 .multidot.x with x=peak area and y=CNPEOC-T
concentration; r=0.9998.
2.3 Results
(a) 51'-O-(2-(2-chloro-6-nitrophenyl)ethoxycarbonyl)thymidine
______________________________________ Irradiation of
5'-O-(2-(2-chloro-6-nitrophenyl)- ethoxycarbonyl)thymidine
______________________________________ Irradiation Time Peak Area
Concentration Relative Quantity (min) CNPEOC-T CNPEOC-T (mM)
CNPEOC-T (%) ______________________________________ 1.00000 458911
0.1489 74.5 2.00000 253292 0.0826 41.3 3.00000 250989 0.0819 40.9
5.00000 84081.0 0.0281 14.0 15.0000 24181.0 0.0088 4.4 30.0000 0 0
0 60.0000 0 0 0 120.000 0 0 0
______________________________________ Irradiation Time Peak Area
Concentration Relative Quantity (min) Thymidine Thymidine (mM)
Thymidine (%) ______________________________________ 1.00000
83460.0 0.0327 16.3 2.00000 221149 0.0858 42.9 3.00000 228893
0.0888 44.4 5.00000 330106 0.1278 63.9 15.0000 432514 0.1673 83.7
30.0000 437588 0.1693 84.6 60.0000 442230 0.1711 85.5 120.000
407543 0.1577 78.9 ______________________________________
This table clearly shows that photolysis across a very large period
follows first-order kinetics, which suggests an unequivocal
cleavage mechanism without yield-decreasing side-reactions.
(b) Further compounds according to the invention
With regard to several other derivatives according to the invention
and to the two comparative compounds V1
(5'-O-(2-nitrobenzyloxycarbonyl)thymidine) and V2
(5'-O-(2.4dinitrobenzyloxycarbonyl)thymidine), the following table
summarizes their results obtained according to the above method and
evaluated in relation to half-life and yield percentage of
deprotected nucleoside derivative, in relation to the maximum
concentration achievable):
______________________________________ Yield Compound No. t.sub.0.5
(min) (% C.sub.max) ______________________________________ 1 2.6 79
2 1.37 92 4 1.71 86 5 1.71 89 6 2.3 70 8 1.68 77 9 7.2 77 10 0.7 89
11 1.46 97 V1 2.5 55 V2 3.3 38
______________________________________
As in evident from the table, the nucleoside derivatives according
to the invention are clearly superior to the protective groups of
the prior art (cf. V1 and V2 )in terms of the photolytic cleavage
of the 5' protective group in relation to rapidity and high yields
(cf. in particular compounds 10 and 11).
2.4 Application example
The compound 5'-O-(2-(2-chloro-6-nitrophenyl) ethoxycarbonyl)
thymidine and other nucleoside derivatives according to the
invention were used according to a method adopted by S.P.A. Fodor
et al. Science 1991, 251, p.767 et seq. for synthesizing
oligonucleotides on a DNA chip. It was shown that the compounds
according to the invention permitted an uncomplicated
oligonucleotide synthesis with very high yields, with the result
that they are suitable in practice for light-controlled parallel
syntheses of oligonucleotides.
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *